Peptide analogues with branched amino acid probe(s)

ABSTRACT

The present invention relates to peptide analogues comprising one or more branched amino acid probes and a peptide, native or variants thereof.

CROSS-REFERENCE

This application is a divisional of U.S. application Ser. No.15/917,223, filed Mar. 9, 2018, which is a divisional of U.S.application Ser. No. 14/693,810, filed Apr. 22, 2015, now issued as U.S.Pat. No. 9,950,027, which application claims the benefit of U.S.Provisional Application No. 61/982,709, filed Apr. 22, 2014, which areherein incorporated by reference in their entirety.

STATEMENT REGARDING SEQUENCE LISTING

The Sequence Listing associated with this application is provided intext format in lieu of a paper copy, and is hereby incorporated byreference into the specification. The name of the text file containingthe Sequence Listing is HOIB_011_03US_ST25.txt. The text file is 40 KB,was created on Sep. 8, 2021, and is being submitted electronically viaEFS-Web.

FIELD OF INVENTION

The present invention relates to branched amino acid probes which areadded to native peptides, or variants of said peptides, to producepeptide analogues or conjugates comprising a peptide and one or morebranched amino acid probes.

BACKGROUND OF THE INVENTION

Proteins and peptides are widely employed for therapeutic purposeswhether in their native forms, variant forms or analogues thereof.Protein therapeutics tend to be specific for their targets, leading topotentially fewer side effects, but often with lower bioavailability,poorer membrane permeability, and metabolic instability, as compared tosmall molecules. Protein-based drugs are generally referred to as‘biologics’ and include molecules such as insulin, growth factors, andengineered antibodies.

Proteinaceous molecules typically require injection; nevertheless,biologics have been an extremely successful class of therapeuticsincluding antibodies for treatment of arthritis and various cancers,soluble proteins for diabetes, myelosuppression and renal anemia; aswell as short injectable peptides for multiple sclerosis, cancers,endometriosis and fibroids and acromegaly.

Peptides represent a class of molecules that have the specificity andpotency of larger protein biologics, but are smaller in size and moreaccessible and cheaper to manufacture using chemical methods, thuspotentially combining some of the advantages of proteins with those ofsmall molecules.

Protein and peptide compounds can be modified in various ways in orderto improve one or more features of the compound, or address one or morepotential draw-backs of the compound. For example, a stabilizing peptidesequence may be added to the N- and/or C-terminus of pharmacologicallyactive peptides potentially making them less susceptible to degradation(WO 99/46283). Further, a linear amino acid probe of 6 amino acidsselected from Lys or Glu added to the N-terminus of α-MSH potentiallyincreases efficacy compared to the native peptide (WO 07/22774). Knownpeptide-drug conjugates further include addition of polycationicpeptides CPP (cell-penetrating peptides) to improve transport across thecell lipid bi-layer.

SUMMARY OF THE INVENTION

The present invention provides peptide analogues comprising a peptide orprotein, native or naturally occurring, or biologically active variantsthereof, and one or more branched amino acid probes (abbreviated BAPherein). Modification of peptides by addition of one or more branchedamino acid probes has not previously been disclosed.

In some embodiments, the peptide analogues provided herein have one ormore improved properties compared to the native peptide. For example, insome embodiments, addition of one or more branched amino acid probes toa peptide potentially improves one or more features of the peptide, suchas

-   -   improve or increase an inherent effect of the peptide (including        for example increasing the activity, affinity and/or efficacy of        a pharmacologically active peptide; improved binding to and/or        activation of one or more relevant receptors);    -   alter an inherent effect of the peptide (including for example        an altered receptor binding profile), or    -   improve or alter an external effect of the peptide (including        for example increased stability, reduced degradation, altered        configuration and/or altered solubility).

Thus, the present invention relates to a peptide analogue comprising apeptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first amino-alkylamino acid residue,said first amino-alkyl amino acid residue optionally being covalentlylinked to a second amino-alkyl amino acid residue, or to a second and athird amino-alkyl amino acid residue, to form a linear chain of 2 or 3amino-alkyl amino acid residues,wherein the side chain(s) of one or more of said first, second and/orthird amino-alkyl amino acid residues are each modified by attaching tothe side chain amino group a molecule independently selected from thegroup consisting of AAA_(q)-AAA; (aa₃)_(p)-AAA_(q); AAA_(q)-(aa₃)_(p);[(aa₃)-AAA]_(p) and [AAA-(aa₃)]p;wherein q is a number selected from 0, 1, 2 and 3; p is a numberselected from 1, 2 and 3; AAA is an amino-alkyl amino acid residue; and(aa₃) is an amino acid residue independently selected from Arg, His, Glyand Ala,wherein said amino-alkyl amino acid residues are optionally acetylated,wherein said first amino-alkyl amino acid residue is covalently linkedto the N-terminus of said peptide, covalently linked to the C-terminusof said peptide, and/or attached to the side chain amino group of anamino-alkyl amino acid residue within said peptide,with the proviso that said branched amino acid probe consists of 2 to 9amino acid residues.

The present invention also encompasses pharmaceutical compositionscomprising the peptide analogues of the present invention, as well asthe analogues of the present invention for use as a medicament.

DESCRIPTION OF DRAWINGS

FIG. 1: Schematic representation of the branched amino acid probeAc-(Ac-Lys-Lys)Lys-, showing the first amino-alkyl amino acid residuebeing a lysine residue (Lys₁) attached to the N-terminus of a peptidesequence via a regular peptide bond, said first lysine being acetylated(COCH₃), and said first lysine modified by attaching to the ε-aminogroup of said first lysine residue two further lysine residues whereinone is also acetylated (the outermost).

DETAILED DESCRIPTION OF THE INVENTION

Peptide Analogues

It is an aspect of the present invention to provide peptide analoguesmodified by addition of one or more branched amino acid probes. Thepeptide analogues may comprise any peptide, polypeptide or protein,native or naturally occurring, or biologically active variants orfragments thereof, which are modified by addition of one or morebranched amino acid probes (abbreviated BAP herein). Thus in oneembodiment the peptide analogues are conjugates comprising a peptidesequence and one or more branched amino acid probes.

The terms ‘peptide analogue’ and ‘protein analogue’ may be usedinterchangeably herein. The terms ‘peptide’ and ‘protein’ may be usedinterchangeably herein. The terms ‘peptide’ and ‘peptide sequence’ maybe used interchangeably herein. The terms ‘peptide sequence’ and ‘aminoacid sequence’ may be used interchangeably herein.

In some embodiments, the peptide analogues provided herein have certainimproved properties, for instance with respect to binding affinityand/or activation of one or more receptors. Still further, in anotherembodiment, the peptide analogues provided herein are more stable, suchas less susceptible to proteases.

It is an aspect of the present invention to provide a peptide analoguecomprising a peptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first amino-alkylamino acid residue,

said first amino-alkyl amino acid residue optionally being covalentlylinked to a second amino-alkyl amino acid residue, or to a second and athird amino-alkyl amino acid residue, to form a linear chain of 2 or 3amino-alkyl amino acid residues,

wherein the side chain(s) of one or more of said first, second and/orthird amino-alkyl amino acid residues are each modified by attaching tothe side chain amino group a molecule independently selected from thegroup consisting of AAA_(q)-AAA; (aa₃)_(p)-AAA_(q); AAA_(q)-(aa₃)_(p);[(aa₃)-AAA]_(p) and [AAA-(aa₃)]_(p);wherein q is a number selected from 0, 1, 2 and 3; p is a numberselected from 1, 2 and 3; AAA is an amino-alkyl amino acid residue; and(aa₃) is an amino acid residue independently selected from Arg, His, Glyand Ala,wherein said amino-alkyl amino acid residues are optionally acetylated,wherein said first amino-alkyl amino acid residue is covalently linkedto the N-terminus of said peptide, covalently linked to the C-terminusof said peptide, and/or attached to the side chain amino group of anamino-alkyl amino acid residue within said peptide,with the proviso that said branched amino acid probe consists of 2 to 9amino acid residues.

According to the invention an amino-alkyl amino acid residue beingcovalently linked to further amino-alkyl amino acid residues and/or apeptide in one embodiment means that a peptide bond is present. In oneembodiment, covalently linked means covalently linked by (a) peptidebond(s). In one embodiment, covalently linked implies that (a) peptidebond(s) is present.

A peptide bond (amide bond) is a covalent chemical bond formed betweentwo molecules when the carboxyl group of one molecule reacts with theamino group of the other molecule, causing the release of a molecule ofH₂O. The process usually occurs between amino acids.

The amino-alkyl amino acid residues (or AAA) according to the inventionmay each be the same (identical) or different (non-identical).

Branched Amino Acid Probe

Amino-Alkyl Amino Acid Residue

According to the present invention an ‘amino-alkyl amino acid residue’(or AAA) is an amino acid having the conventional amine (—NH₂) andcarboxylic acid (—COOH) functional groups, and a side chain attached tothe first (alpha-) carbon atom, wherein the side-chain comprises analkyl group (—C_(n)H_(2n+1)) and an amino group (NH₂); in one embodimentthe side chain comprises an amino-alkyl group (—C_(n)H_(2n)NH₂).

Thus an amino-alkyl amino acid residue (or AAA) is an amino acid with aside chain comprising or consisting of an amino-alkyl group(—C_(n)H_(2n)NH₂), in one embodiment denoted a side chain amino-alkylgroup.

In one embodiment the side chain alkyl group is derived from the groupconsisting of methyl (CH₃—), ethyl (C₂H₅—), propyl (C₃H₇—), butyl(C₄H₉—), pentyl (C₅H₁₁—), hexyl (C₆H₁₃—), heptyl (C₇H₁₅—), octyl(C₈H₁₇—), nonyl (C₉H₁₉—), decyl (C₁₀H₂₁—), undecyl (C₁₁H₂₃—) and dodecyl(C₁₂H₂₅—).

In one embodiment the side chain amino group (NH₂) of said amino-alkylamino acid residue is the amine of methylamine, the amine of ethylamine,the amine of propylamine, the amine of n-butylamine, the amine ofpentylamine, the amine of n-hexylamine, the amine of heptylamine, theamine of octylamine, the amine of nonylamine, the amine of decylamine,the amine of undecylamine or the amine of dodecylamine.

In one embodiment the side chain amino-alkyl group according to theinvention is selected from the group consisting of methylamine(—CH₂NH₂), ethylamine (—C₂H₄NH₂), propylamine (—C₃H₆NH₂), n-butylamine(—C₄H₈NH₂), pentylamine (—C₅H₁₀NH₂), n-hexylamine (—C₆H₁₂NH₂),heptylamine (—C₇H₁₄NH₂), octylamine (—C₈H₁₆NH₂), nonylamine (—C₉H₁₈NH₂),decylamine (—C₁₀H₂₀NH₂), undecylamine (—C₁₁H₂₂NH₂) and dodecylamine(—C₁₂H₂₄NH₂).

In one embodiment the side chain amino group (NH₂) of said first, secondand/or third amino-alkyl amino acid residues are each modified byattaching a molecule thereto.

In one embodiment the side chain amino group of said amino-alkyl aminoacid residue is selected from the group consisting of

the β (beta) amino group (1 methylene in the side chain; methylamine);the γ (gamma) amino group (2 methylenes in the side chain, ethylamine);the δ (delta) amino group (3 methylenes in the side chain, propylamine);=ornithinethe ε (epsilon) amino group (4 methylenes in the side chain;n-butylamine); =lysinethe ζ (zeta) amino group (5 methylenes in the side chain; pentylamine);the η (eta) amino group (6 methylenes in the side chain; n-hexylamine);the θ (theta) amino group (7 methylenes in the side chain; heptylamine);the ι (iota) amino group (8 methylenes in the side chain; octylamine);the κ (kappa) amino group (9 methylenes in the side chain; nonylamine);the λ (lambda) amino group (10 methylenes in the side chain;decylamine);the μ (mu) amino group (11 methylenes in the side chain; undecylamine);andthe ν (nu) amino group (12 methylenes in the side chain; dodecylamine).

For example, the ε-amino group is attached to the fifth carbon beginningfrom (including) the α-carbon, which α-carbon is attached to thecarboxyl (C═OOH) group.

An amino-alkyl amino acid residue wherein the side chain is n-butylamineand the side chain amino group is the ε (epsilon) amino group is lysine(Lys, K).

Likewise, the δ-amino group is attached to the fourth carbon beginningfrom the α-carbon.

An amino-alkyl amino acid residue wherein the side chain is propylamineand the side chain amino group is the δ (delta) amino group is ornithine(Orn).

Ornithine is formed in cells by deguanidation of arginine. While it isnot used in proteinogenesis in vivo it is a participant in severalenzyme pathways and appears to play a role in nitrogen balance in vivoas it can be gaunidated enzymatically to form arginine.

Any amino acid according to the present invention may be in the L- orD-configuration. If nothing is specified, reference to the L-isomericform is preferably meant.

It follows that the amino-alkyl amino acid residues of the invention inone embodiment are individually in the L- or D-configuration. In oneembodiment the amino-alkyl amino acid residues are in theL-configuration.

In one embodiment the amino-alkyl amino acid residues of the inventionare individually selected from the group consisting of lysine andornithine.

In one embodiment the amino-alkyl amino acid residues of the inventionare individually selected from the group consisting of L-lysine,D-lysine, L-ornithine and D-ornithine.

In one embodiment the amino-alkyl amino acid residues of the inventionare selected from the group consisting of L-lysine and L-ornithine.

In one embodiment the amino-alkyl amino acid residues of the inventionare selected from the group consisting of L-lysine and D-lysine.

In one embodiment the amino-alkyl amino acid residues of the inventionare selected from the group consisting L-ornithine and D-ornithine.

In one embodiment there is provided a peptide analogue comprising apeptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first amino acidresidue selected from lysine and ornithine,said first amino acid residue optionally being covalently linked to asecond, or to a second and a third amino acid residue selected fromlysine or ornithine, to form a linear chain of 2 or 3 lysine orornithine residues,wherein the side chain(s) of one or more of said first, second and/orthird lysine or ornithine residues are modified by attaching to theδ-amino group (ornithine) or the ε-amino group a molecule independentlyselected from the group consisting ofLys_(q)-Lys; (aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p);[Lys-(aa₃)]_(p);Orn_(q)-Orn; (aa₃)_(p)-Orn_(q); Orn_(q)-(aa₃)_(p); [(aa₃)-Orn]_(p) and[Orn-(aa₃)]_(p);Orn_(p)-Lys_(p); Lys_(p)-Orn_(p); [Orn-Lys]_(p) and [Lys-Orn]_(p);wherein q is a number selected from 0, 1, 2 and 3; p is a numberselected from 1, 2 and 3; and (aa₃) is an amino acid residueindependently selected from Arg, His, Gly and Ala,wherein said lysine and ornithine residues are optionally acetylated,wherein said lysine and ornithine residues are in the L- orD-configuration,wherein said first lysine or ornithine residue is covalently linked tothe N-terminus of said peptide,covalently linked to the C-terminus of said peptide, and/or attached tothe ε-amino group of a lysine residue or the δ-amino group of anornithine residue within said peptide, with the proviso that saidbranched amino acid probe consists of 2 to 9 amino acid residues.

In one embodiment there is provided a peptide analogue comprising apeptide and one or more branched amino acid probes,

wherein said branched amino acid probe comprises a first lysine residue,said first lysine residue optionally being covalently linked to asecond, or to a second and a third lysine residue, to form a linearchain of 2 or 3 lysine residues,wherein the side chain(s) of one or more of said first, second and/orthird lysine residues are modified by attaching to the ε-amino group amolecule independently selected from the group consisting ofLys_(q)-Lys; (aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p);[Lys-(aa₃)]_(p); wherein q is a number selected from 0, 1, 2 and 3; p isa number selected from 1, 2 and 3; and (aa₃) is an amino acid residueindependently selected from Arg, His, Gly and Ala,wherein said lysine residues are optionally acetylated,wherein said lysine residues are in the L- or D-configuration,wherein said first lysine residue is covalently linked to the N-terminusof said peptide, covalently linked to the C-terminus of said peptide,and/or attached to the ε-amino group of a lysine residue within saidpeptide,with the proviso that said branched amino acid probe consists of 2 to 9amino acid residues.

Branching the Probe

A branched amino acid probe according to the present invention in oneembodiment consists of 2 to 9 amino acid residues.

In one embodiment the branched amino acid probe consist of from 2 to 3amino acid residues, such as from 3 to 4 amino acid residues, forexample from 4 to 5 amino acid residues, such as from 5 to 6 amino acidresidues, for example from 6 to 7 amino acid residues, such as from 7 to8 amino acid residues, for example from 8 to 9 amino acid residues.

In one embodiment the branched amino acid probe consist of 2 amino acidresidues, such as 3 amino acid residues, for example 4 amino acidresidues, such as 5 amino acid residues, for example 6 amino acidresidues, such as 7 amino acid residues, for example 8 amino acidresidues, such as 9 amino acid residues.

In one embodiment the branched amino acid probe comprises a firstamino-alkyl amino acid residue (also denoted AAA₁), which firstamino-alkyl amino acid residue is connected to a peptide to provide apeptide analogue according to the invention.

In one embodiment the first amino-alkyl amino acid of (each of) the oneor more branched amino acid probe(s) is (are) covalently linked to theN-terminus of said peptide, covalently linked to the C-terminus of saidpeptide, and/or attached to the side chain amino group of an amino-alkylamino acid residue within said peptide.

In one embodiment the first amino-alkyl amino acid of the branched aminoacid probe is acetylated.

In one embodiment said first amino-alkyl amino acid residue iscovalently linked to a second amino-alkyl amino acid residue to form alinear chain of 2 amino-alkyl amino acid residues.

In one embodiment the second amino-alkyl amino acid of the branchedamino acid probe is acetylated.

In one embodiment said first amino-alkyl amino acid residue iscovalently linked to a second and (covalently linked to) a thirdamino-alkyl amino acid residue to form a linear chain of 3 amino-alkylamino acid residues. In this setting, it is understood that the firstamino-alkyl amino acid residue may have the second and third amino-alkylamino acid residues both attached at its amine group or both attached toits carboxylic acid group; or it may have the second amino-alkyl aminoacid residue attached at its amine group and the third amino-alkyl aminoacid residue attached at its carboxylic acid group.

In one embodiment the third amino-alkyl amino acid of the branched aminoacid probe is acetylated.

The second and third amino-alkyl amino acid residues may be denoted AAA₂and AAA₃, respectively.

In one embodiment each of said first, second and/or third amino-alkylamino acid residues is an amino acid having a side chain amino-alkylgroup selected from the group consisting of methylamine (—CH₂NH₂),ethylamine (—C₂H₄NH₂), propylamine (—C₃H₆NH₂), n-butylamine (—C₄H₈NH₂),pentylamine (—C₅H₁₀NH₂), n-hexylamine (—C₆H₁₂NH₂), heptylamine(—C₇H₁₄NH₂), octylamine (—C₈H₁₆NH₂), nonylamine (—C₉H₁₈NH₂), decylamine(—C₁₀H₂₀NH₂), undecylamine (—C₁₁H₂₂NH₂) and dodecylamine (—C₁₂H₂₄NH₂).

In one embodiment each of the first, second and/or third amino-alkylamino acids of the branched amino acid probe are individually selectedfrom the group consisting of L-lysine, D-lysine, L-ornithine andD-ornithine.

In one embodiment each of the first, second and third amino-alkyl aminoacids of the branched amino acid probe are lysine residues (includingL-lysine and D-lysine).

In one embodiment each of the first, second and third amino-alkyl aminoacids of the branched amino acid probe are acetylated (Ac-AAA) (COCH₃).

In one embodiment, the first, the first and second, and/or the first,second and third amino-alkyl amino acid residues of the branched aminoacid probe are referred to as the amino-alkyl amino acid backbone of thebranched amino acid probe (AAA₁, AAA₁₋₂, AAA₁₋₃).

In one embodiment the first, second and third amino-alkyl amino acidresidues are lysine residues. In one embodiment the first, the first andsecond, and/or the first, second and third lysine residues of thebranched amino acid probe are referred to as the lysine backbone of thebranched amino acid probe (Lys₁, Lys₁₋₂, Lys₁₋₃).

In one embodiment the first lysine residue, or the second lysineresidue, or the third lysine residue, or the first and the second lysineresidues, or the first and the third lysine residues, or the second andthe third lysine residues, or the first, the second and the third lysineresidues of the lysine backbone of the branched amino acid probe areacetylated (Ac-Lys).

In one embodiment each of the first, second and third lysine residues ofthe branched amino acid probe are acetylated (Ac-Lys).

In one embodiment the side chain(s) of one or more of each of saidfirst, second and/or third amino-alkyl amino acid residues are modifiedby attaching to the side chain amino group a molecule independentlyselected from the group consisting of AAA_(q)-AAA; (aa₃)_(p)-AAA_(q);AAA_(q)-(aa₃)_(p); [(aa₃)-AAA]_(p) and [AAA-(aa₃)]_(p); wherein q is anumber selected from 0, 1, 2 and 3; p is a number selected from 1, 2 and3; AAA is an amino-alkyl amino acid residue; and (aa₃) is an amino acidresidue independently selected from Arg, His, Gly and Ala.

In one embodiment the side chain(s) of one or more of each of saidfirst, second and/or third amino-alkyl amino acid residues are modifiedby attaching to the side chain amino group a molecule independentlyselected from the group consisting of

Lys_(q)-Lys; (aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p);[Lys-(aa₃)]_(p);Orn_(q)-Orn; (aa₃)_(p)-Orn_(q); Orn_(q)-(aa₃)_(p); [(aa₃)-Orn]_(p) and[Orn-(aa₃)]_(p);Orn_(p)-Lys_(p); Lys_(p)-Orn_(p); [Orn-Lys]_(p) and [Lys-Orn]_(p);wherein q is a number selected from 0, 1, 2 and 3; p is a numberselected from 1, 2 and 3; and (aa₃) is an amino acid residueindependently selected from Arg, His, Gly and Ala.

In one embodiment the side chain(s) of one or more of each of saidfirst, second and/or third amino-alkyl amino acid residues are modifiedby attaching to the side chain amino group a molecule independentlyselected from the group consisting of Lys_(q)-Lys; (aa₃)_(p)-Lys_(q);Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p) and [Lys-(aa₃)]_(p); wherein q is anumber selected from 0, 1, 2 and 3; p is a number selected from 1, 2 and3; Lys is a lysine residue selected from L-Lys and D-Lys; and (aa₃) isan amino acid residue independently selected from Arg, His, Gly and Ala.

In one embodiment the side chain(s) of one or more of each of saidfirst, second and/or third lysine residues of the lysine backbone aremodified by attaching to the ε-amino group of the side chain a moleculeindependently selected from the group consisting of Lys_(q)-Lys;(aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p) and[Lys-(aa₃)]_(p); wherein q is a number selected from 0, 1, 2 and 3; p isa number selected from 1, 2 and 3; Lys is a lysine residue selected fromL-Lys and D-Lys; and (aa₃) is an amino acid residue independentlyselected from Arg, His, Gly and Ala.

In one embodiment the side chain(s) of one or more of each of saidfirst, second and/or third lysine residues of the lysine backbone aremodified by attaching to the ε-amino group of the side chain a moleculebeing Lys_(q)-Lys; wherein q is a number selected from 0, 1, 2 and 3 andLys is a lysine residue selected from L-Lys and D-Lys.

In one embodiment the side chain of i) one of said first, second and/orthird amino-alkyl amino acid residues, ii) two of said first, secondand/or third amino-alkyl amino acid residues, or iii) all three of thefirst, second and third amino-alkyl amino acid residues, are modified byattaching to the side chain amino group a molecule as defined herein.

In one embodiment the side chain of i) the first amino-alkyl amino acidresidue, ii) the second amino-alkyl amino acid residue, iii) the thirdamino-alkyl amino acid residue, iv) the first and the second amino-alkylamino acid residues, v) the first and the third amino-alkyl amino acidresidues, vi) the second and the third amino-alkyl amino acid residues,or vii) the first, the second and the third amino-alkyl amino acidresidues, are each modified by attaching to the side chain amino group amolecule as defined herein.

In one embodiment the first lysine residue, or the second lysineresidue, or the third lysine residue, or the first and the second lysineresidues, or the first and the third lysine residues, or the second andthe third lysine residues, or the first, the second and the third lysineresidues of the lysine backbone of the branched amino acid probe of theinvention are each modified by attaching a molecule to the ε-aminogroup.

In one embodiment, the molecule to be attached to the ε-amino group(s)of the one or more lysine residues of the lysine backbone of thebranched amino acid probe are independently selected from the groupconsisting of Lys_(q)-Lys; (aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p);[(aa₃)-Lys]_(p) and [Lys-(aa₃)]_(p), wherein q is a number selected from0, 1, 2 and 3; p is a number selected from 1, 2 and 3, and (aa₃) is anamino acid residue independently selected from Arg, His, Gly and Ala.

It follows that in one embodiment the first lysine residue, or thesecond lysine residue, or the third lysine residue, or the first and thesecond lysine residues, or the first and the third lysine residues, orthe second and the third lysine residues, or the first, the second andthe third lysine residues of the branched amino acid probe are modifiedby attaching to the ε-amino group(s) a molecule independently selectedfrom the group consisting of Lys_(q)-Lys; (aa₃)_(p)-Lys_(q);Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p) and [Lys-(aa₃)]_(p), wherein q is anumber selected from 0, 1, 2 and 3; p is a number selected from 1, 2 and3, and (aa₃) is an amino acid residue independently selected from Arg,His, Gly and Ala.

In a particular embodiment (aa₃) is an amino acid residue independentlyselected from Gly and Ala. In a further embodiment, (aa₃) is Gly.

In one embodiment, the molecules to be attached to the side chain aminogroup(s) are further acetylated. In one embodiment the molecules areindependently selected from the group consisting of Ac-AAA_(q)-AAA;Ac-(aa₃)_(p)-AAA_(q); Ac-AAA_(q)-(aa₃)_(p); Ac-[(aa₃)-AAA]_(p) andAc-[AAA-(aa₃)]_(p), and/or AAA_(q)-AAAs; (aa₃)_(p)-AAA_(q);AAA_(q)-(aa₃)_(p); [(aa₃)-AAA]_(p) and [AAA-(aa₃)]_(p).

In one embodiment the molecules are independently selected from thegroup consisting of Ac-Orn_(q)-Orn; Ac-(aa₃)_(p)-Orn_(q);Ac-Orn_(q)-(aa₃)_(p); Ac-[(aa₃)-Orn]_(p); Ac-[Orn-(aa₃)]_(p);Ac-Orn_(p)-Lys_(p); Ac-Lys_(p)-Orn_(p); Ac-[Orn-Lys]_(p) andAc-[Lys-Orn]_(p), and/or Orn_(q)-Orn; (aa₃)_(p)-Orn_(q);Orn_(q)-(aa₃)_(p); [(aa₃)-Orn]_(p) and [Orn-(aa₃)]_(p); Orn_(p)-Lys_(p);Lys_(p)-Orn_(p); [Orn-Lys]_(p) and [Lys-Orn]_(p).

It follows that the molecules are in one embodiment independentlyselected from the group consisting of Ac-Lys_(q)-Lys;Ac-(aa₃)_(p)-Lys_(q); Ac-Lys_(q)-(aa₃)_(p); Ac-[(aa₃)-Lys]_(p) andAc-[Lys-(aa₃)]_(p), and/or Lys_(q)-Lys; (aa₃)_(p)-Lys_(q);Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p) and [Lys-(aa₃)]_(p).

In a particular embodiment, the molecule to be attached to the sidechain amino group(s) is AAA_(q)-AAA, wherein q is a number selected from0, 1, 2 and 3.

It follows that in one embodiment the branched amino acid probe consistsof 2 to 9 amino-alkyl amino acid residues. In one embodiment said 2 to 9amino-alkyl amino acid residues are individually selected from the groupconsisting of L-lysine, D-lysine, L-ornithine and D-ornithine.

In a particular embodiment, the molecule to be attached to the sidechain amino group(s) is Lys_(q)-Lys, wherein q is a number selected from0, 1, 2 and 3.

It follows that in one embodiment the branched amino acid probe of theinvention consists of 2 to 9 lysine residues selected from Lys andD-Lys.

In one embodiment, the branched amino acid probe comprises a maximum of1, 2, 3 or 4 amino acids selected from Arg, His, Gly and Ala (aa₃),wherein the remaining amino acids are amino-alkyl amino acid residues.In another embodiment, the branched amino acid probe comprises a maximumof 1 Arg residue, and/or comprises a maximum of 1 His residue, and/orcomprises a maximum of 1 Gly residue, and/or comprises a maximum of 1Ala residue.

In one embodiment, the molecule to be attached to the side chain aminogroup(s) of one or more of the first, second and/or third amino-alkylamino acid residues is selected from the group consisting of AAA,Ac-AAA, AAA-AAA, Ac-AAA-AAA, AAA-AAA-AAA, Ac-AAA-AAA-AAA,AAA-AAA-AAA-AAA, Ac-AAA-AAA-AAA-AAA, AAA-Gly-AAA, Ac-AAA-Gly-AAA,AAA-AAA-Gly, Ac-AAA-AAA-Gly, AAA-Gly, Ac-AAA-Gly, AAA-Ala-AAA,Ac-AAA-Ala-AAA, AAA-AAA-Ala, Ac-AAA-AAA-Ala, AAA-Ala, Ac-AAA-Ala,AAA-His-AAA, Ac-AAA-His-AAA, AAA-AAA-His, Ac-AAA-AAA-His, AAA-His,Ac-AAA-His, AAA-Arg-AAA, Ac-AAA-Arg-AAA, AAA-AAA-Arg, Ac-AAA-AAA-Arg,AAA-Arg and Ac-AAA-Arg; wherein AAA is an amino-alkyl amino acid residueas specified herein (optionally individually acetylated). Theabove-mentioned AAA, Gly, Ala, His and Arg amino acid residues may eachbe in the L- or D-conformation.

In one embodiment, the molecule to be attached to the side chain aminogroup(s) of one or more of the first, second and/or third amino-alkylamino acid residues is selected from the group consisting of Lys,Ac-Lys, Lys-Lys, Ac-Lys-Lys, Lys-Lys-Lys, Ac-Lys-Lys-Lys,Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys-Gly-Lys,Lys-Lys-Gly, Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys,Ac-Lys-Ala-Lys, Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala,Lys-His-Lys, Ac-Lys-His-Lys, Lys-Lys-His, Ac-Lys-Lys-His, Lys-His,Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac-Lys-Lys-Arg,Lys-Arg and Ac-Lys-Arg.

In a particular embodiment, the molecule to be attached to the ε-aminogroup(s) of one or more of the first, second and/or third lysineresidues is selected from the group consisting of Lys, Ac-Lys, Lys-Lys,Ac-Lys-Lys, Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys,Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys-Gly-Lys, Lys-Lys-Gly,Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala-Lys,Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys,Ac-Lys-His-Lys, Lys-Lys-His, Ac-Lys-Lys-His, Lys-His, Ac-Lys-His,Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg andAc-Lys-Arg.

In a particular embodiment, the branched amino acid probe of theinvention comprise or consist of a first lysine residue selected fromLys and D-Lys, said first lysine residue being optionally acetylated,wherein said first lysine residue is modified by attaching to theε-amino group of said first lysine residue a molecule selected from thegroup consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys, Lys-Lys-Lys,Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys,Ac-Lys-Gly-Lys, Lys-Lys-Gly, Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly,Lys-Ala-Lys, Ac-Lys-Ala-Lys, Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala,Ac-Lys-Ala, Lys-His-Lys, Ac-Lys-His-Lys, Lys-Lys-His, Ac-Lys-Lys-His,Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg,Ac-Lys-Lys-Arg, Lys-Arg and Ac-Lys-Arg.

In a particular embodiment, the branched amino acid probe of theinvention comprise or consist of a first and a second lysine residueeach selected from Lys and D-Lys, said first and second lysine residueseach being optionally acetylated, wherein i) said first lysine residue,ii) said second lysine residue, or iii) said first and second residueare each modified by attaching to the C-amino group of said lysineresidue a molecule selected from the group consisting of Lys, Ac-Lys,Lys-Lys, Ac-Lys-Lys, Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys,Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys-Gly-Lys, Lys-Lys-Gly,Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala-Lys,Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys,Ac-Lys-His-Lys, Lys-Lys-His, Ac-Lys-Lys-His, Lys-His, Ac-Lys-His,Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg andAc-Lys-Arg.

In a particular embodiment, the branched amino acid probe of theinvention comprise or consist of a first, a second and a third lysineresidue each selected from Lys and D-Lys, said first, second and thirdlysine residue each being optionally acetylated, wherein i) said firstlysine residue, ii) said second lysine residue, iii) said third lysineresidue, iv) said first and second lysine residue, v) said first andthird lysine residue, vi) said second and third lysine residue, or vii)said first, second and third lysine residues are each modified byattaching to the ε-amino group of said lysine residue a moleculeselected from the group consisting of Lys, Ac-Lys, Lys-Lys, Ac-Lys-Lys,Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys, Ac-Lys-Lys-Lys-Lys,Lys-Gly-Lys, Ac-Lys-Gly-Lys, Lys-Lys-Gly, Ac-Lys-Lys-Gly, Lys-Gly,Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala-Lys, Lys-Lys-Ala, Ac-Lys-Lys-Ala,Lys-Ala, Ac-Lys-Ala, Lys-His-Lys, Ac-Lys-His-Lys, Lys-Lys-His,Ac-Lys-Lys-His, Lys-His, Ac-Lys-His, Lys-Arg-Lys, Ac-Lys-Arg-Lys,Lys-Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg and Ac-Lys-Arg.

In one embodiment the branched amino acid probe comprises or consists ofthe formula: Ac-(Ac-Lys-Lys)Lys₁-(identical to Ac-(Ac-Lys-Lys)Lys-),wherein Lys₁ is the first lysine residue, which is acetylated, and(Ac-Lys-Lys) is the molecule attached to the ε-amino group of said firstlysine residue Lys₁. FIG. 1 illustrates this formula/structure.

In one embodiment the branched amino acid probe comprises or consists ofa formula selected from the group consisting of Ac-(Ac-Lys)Lys₁-,Ac-(Ac-Lys-Lys)Lys₁-, Ac-(Ac-Lys-Lys-Lys)Lys₁-,Ac-(Ac-Lys-Lys-Lys-Lys)Lys₁-, Ac-(Ac-Lys-Gly-Lys)Lys₁-,Ac-(Ac-Lys-Lys-Gly)Lys₁-, Ac-(Ac-Lys-Gly)Lys₁-,Ac-(Ac-Lys-Ala-Lys)Lys₁-, Ac-(Ac-Lys-Lys-Ala)Lys₁-,Ac-(Ac-Lys-Ala)Lys₁-, Ac-(Ac-Lys-His-Lys)Lys₁-,Ac-(Ac-Lys-Lys-His)Lys₁-, Ac-(Ac-Lys-His)Lys₁-,Ac-(Ac-Lys-Arg-Lys)Lys₁-, Ac-(Ac-Lys-Lys-Arg)Lys₁-, andAc-(Ac-Lys-Arg)Lys₁-.

More specifically, in one embodiment the branched amino acid probecomprises or consists of a formula selected from the group consisting ofAc-(Ac-Lys)Lys₁-, Ac-(Ac-Lys-Lys)Lys₁-, Ac-(Ac-Lys-Lys-Lys)Lys₁-,Ac-(Ac-Lys-Lys-Lys-Lys)Lys₁-, Ac-(Ac-Lys-Gly-Lys)Lys₁-,Ac-(Ac-Lys-Lys-Gly)Lys₁- and Ac-(Ac-Lys-Gly)Lys₁-.

In one embodiment the branched amino acid probe comprises or consists ofthe formula: Ac-(Ac-Lys)Lys₂-Lys₁-, wherein Lys₁ is the first lysineresidue, which is acetylated, Lys₂ is the second lysine residue beingattached to Lys₁ via a peptide bond, and (Ac-Lys) is the moleculeattached to the ε-amino group of said first lysine residue Lys₁.

In one embodiment the branched amino acid probe comprises or consists ofthe formula: Ac-Lys₂-(Ac-Lys)Lys₁-, wherein the molecule (Ac-Lys) isattached to the ε-amino group of said second lysine residue Lys₂.

In one embodiment the branched amino acid probe(s) is selected from thegroup consisting of Ac-(Ac-Lys)Lys-Lys-, (Ac-Lys)Lys-Lys-,Ac-(Lys)Lys-Lys-, (Lys)Lys-Lys-; Ac-Lys-(Ac-Lys)Lys-, Lys-(Ac-Lys)Lys-,Ac-Lys-(Lys)Lys-, Lys-(Lys)Lys-; Ac-(Ac-Lys-Lys)-Lys-,(Ac-Lys-Lys)-Lys-, Ac-(Lys-Lys)-Lys- and (Lys-Lys)-Lys-.

In one embodiment the branched amino acid probe(s) is selected from thegroup consisting of Ac-(Ac-Lys)Lys-, Ac-(Lys)Lys- and (Lys)Lys-.

In one embodiment the branched amino acid probe is selected from thegroup consisting of Ac-(Ac-Lys)Lys₂-Lys₁-, Ac-(Ac-Lys-Lys)Lys₂-Lys₁-,Ac-(Ac-Lys-Gly)Lys₂-Lys₁-, Ac-(Ac-Lys-Lys-Lys)Lys₂-Lys₁-,Ac-(Ac-Lys-Lys-Lys-Lys)Lys₂-Lys₁-, Ac-Lys₂-(Ac-Lys)-Lys₁-,Ac-Lys₂-(Ac-Lys-Lys)-Lys₁-, Ac-Lys₂-(Ac-Lys-Gly)-Lys₁-,Ac-Lys₂-(Ac-Lys-Lys-Lys)-Lys₁-, Ac-Lys₂-(Ac-Lys-Lys-Lys-Lys)-Lys₁-,Ac-(Ac-Lys)Lys₂-(Ac-Lys-)-Lys₁-, Ac-(Ac-Lys)Lys₂-(Ac-Lys-Lys-)-Lys₁-,and Ac-(Ac-Lys-Lys)Lys₂-(Ac-Lys-Lys-)-Lys₁-.

More specifically, in one embodiment the branched amino acid probe isselected from the group consisting of Ac-(Ac-Lys)Lys₂-Lys₁-,Ac-(Ac-Lys-Lys)Lys₂-Lys₁-, Ac-(Ac-Lys-Gly)Lys₂-Lys₁-,Ac-Lys₂-(Ac-Lys)-Lys₁-, Ac-Lys₂-(Ac-Lys-Lys)-Lys₁-,Ac-Lys₂-(Ac-Lys-Gly)-Lys₁-, Ac-(Ac-Lys)Lys₂-(Ac-Lys-)-Lys₁-,Ac-(Ac-Lys)Lys₂-(Ac-Lys-Lys-)-Lys₁-, andAc-(Ac-Lys-Lys)Lys₂-(Ac-Lys-Lys-)-Lys₁-.

In one embodiment the branched amino acid probe is selected from thegroup consisting of Ac-Lys₃-Lys₂-(Ac-Lys)Lys₁-,Ac-Lys₃-(Ac-Lys)Lys₂-Lys₁-, Ac-(Ac-Lys)Lys₃-Lys₂-Lys₁-,Ac-Lys₃-(Ac-Lys)Lys₂-(Ac-Lys)Lys₁-, Ac-(Ac-Lys)Lys₃-(Ac-Lys)Lys₂-Lys₁-,and Ac-(Ac-Lys)Lys₃-Lys₂-(Ac-Lys)Lys₁-.

In a particular embodiment the branched amino acid probe is selectedfrom the group consisting of Ac-(Ac-Lys)Lys₁-, Ac-(Ac-Lys-Lys)Lys₁-,Ac-(Ac-Lys-Lys-Lys)Lys₁-, Ac-(Ac-Lys-Lys-Lys-Lys)Lys₁-,Ac-(Ac-Lys-Gly-Lys)Lys₁-, Ac-(Ac-Lys-Lys-Gly)Lys₁-,Ac-(Ac-Lys-Gly)Lys₁-, Ac-(Ac-Lys)Lys₂-Lys₁-, Ac-(Ac-Lys-Lys)Lys₂-Lys₁-,Ac-(Ac-Lys-Gly)Lys₂-Lys₁-, Ac-Lys₂-(Ac-Lys)-Lys₁-,Ac-Lyse-(Ac-Lys-Lys)-Lys₁-, Ac-Lyse-(Ac-Lys-Gly)-Lys₁-,Ac-(Ac-Lys)Lys₂-(Ac-Lys-)-Lys₁-, Ac-(Ac-Lys)Lys₂-(Ac-Lys-Lys-)-Lys₁-,Ac-(Ac-Lys-Lys)Lys₂-(Ac-Lys-Lys-)-Lys₁-, Ac-Lys₃-Lys₂-(Ac-Lys)Lys₁-,Ac-Lys₃-(Ac-Lys)Lys₂-Lys₁-, Ac-(Ac-Lys)Lys₃-Lys₂-Lys₁-,Ac-Lys₃-(Ac-Lys)Lys₂-(Ac-Lys)Lys₁-, Ac-(Ac-Lys)Lys₃-(Ac-Lys)Lys₂-Lys₁-,and Ac-(Ac-Lys)Lys₃-Lys₂-(Ac-Lys)Lys₁-.

In a particular embodiment, the branched amino acid probe consists of 3lysine residues (selected from L-Lys and D-Lys).

In another embodiment, the branched amino acid probe consists of 2lysine residues (selected from L-Lys and D-Lys).

In a particular embodiment, the branched amino acid probe consists of afirst and a second lysine residue selected from Lys and D-Lys, whereinone or both of the first and second lysine residues are modified byattaching to the ε-amino group of said first and/or second lysineresidue one lysine residue selected from Lys and D-Lys; wherein each ofsaid lysine residues are optionally acetylated.

In a particular embodiment, the branched amino acid probe consists of afirst lysine residue selected from Lys and D-Lys, wherein said firstlysine residue is modified by attaching to the ε-amino group of saidfirst lysine residue two lysine residues selected from Lys and D-Lys;wherein each of said lysine residues are optionally acetylated.

Linking the Branched Amino Acid Probes and the Peptide

According to the invention, the first amino-alkyl amino acid residue ofeach of the one or more branched amino acid probes is covalently linkedto the N-terminus of a peptide, covalently linked to the C-terminus of apeptide, and/or attached to the side chain amino group of an amino-alkylamino acid residue within a peptide to be modified according to theinvention.

Attaching one or more branched amino acid probes to a peptide yields apeptide/probe-conjugate.

It is understood that covalently bound or linked to the N-terminus of apeptide means that a branched amino acid probe of the invention islinked by a peptide bond between the most N-terminal amino acid of saidpeptide and the first amino-alkyl amino acid residue of the branchedamino acid probe.

Likewise, it is understood that covalently bound or linked to theC-terminus of a peptide means that a branched amino acid probe of theinvention is linked by a peptide bond between the most C-terminal aminoacid of said peptide and the first amino-alkyl amino acid residue of thebranched amino acid probe.

Furthermore, it is understood that a branched amino acid probe in oneembodiment is attached to the side chain amino group of an amino-alkylamino acid residue within said peptide sequence.

In one particular embodiment said amino-alkyl amino acid residue withinsaid peptide sequence is selected from the group consisting of anornithine residue and a lysine residue. In one particular embodimentsaid amino-alkyl amino acid residue within said peptide sequence is alysine residue.

In one embodiment the first amino-alkyl amino acid residue of thebranched amino acid probe of the invention is attached to the δ-aminogroup of an ornithine residue within said peptide or the ε-amino groupof a lysine residue within said peptide.

In one embodiment the first amino-alkyl amino acid residue of thebranched amino acid probe of the invention is attached to the ε-aminogroup of a lysine residue within said peptide.

It is understood that an amino-alkyl amino acid residue within saidpeptide sequence means that the amino-alkyl amino acid residue does notform part of the branched amino acid probe per se, but is a residueoccurring within the existing amino acid sequence of the peptide. Saidamino-alkyl amino acid residue can be positioned at any position of thepeptide, i.e. it may be the first amino acid of the peptide, the secondamino acid of the peptide, the third amino acid of the peptide, thefourth amino acid of the peptide, and so forth continuing until the lastamino acid residue of the peptide.

According to the invention, a peptide analogue comprising one or morebranched amino acid probes means that the peptide analogue in oneembodiment comprises 1 branched amino acid probe, such as 2 branchedamino acid probes, for example 3 branched amino acid probes, such as 4branched amino acid probes, for example 5 branched amino acid probes,such as 6 branched amino acid probes, for example 7 branched amino acidprobes, such as 8 branched amino acid probes, for example 9 branchedamino acid probes, such as 10 branched amino acid probes.

In principle the peptide analogue can comprise any number of branchedamino acid probes provided they can be attached to the peptide(N-terminally, C-terminally and/or one or more amino-alkyl amino acidresidues within said peptide sequence.

In one embodiment of the invention the peptide analogue of the inventioncomprises 1 branched amino acid probe.

In one embodiment the peptide analogue comprises 1 branched amino acidprobe, which branched amino acid probe is covalently bound to theN-terminus of the peptide.

In one embodiment the peptide analogue comprises 1 branched amino acidprobe, which branched amino acid probe is covalently bound to theC-terminus of the peptide.

In one embodiment the peptide analogue comprises 1 branched amino acidprobe, which branched amino acid probe is attached to the side chainamino group of an amino-alkyl amino acid residue within said peptidesequence.

In one embodiment the peptide analogue comprises more than one (two ormore) branched amino acid probe(s). In the embodiments wherein thepeptide analogue comprises more than one branched amino acid probe it isunderstood that the more than one branched amino acid probes mayindividually be the same (identical) or different (non-identical).

In one embodiment of the invention the peptide analogue of the inventioncomprises 2 branched amino acid probes.

In one embodiment the peptide analogue comprises 2 branched amino acidprobes, wherein one branched amino acid probe is covalently bound to theN-terminus of the peptide and another branched amino acid probe iscovalently bound to the C-terminus of the peptide.

In one embodiment the peptide analogue comprises 2 branched amino acidprobes, wherein one branched amino acid probe is covalently bound to theN-terminus of the peptide and another branched amino acid probe isattached to the side chain amino group of an amino-alkyl amino acidresidue within said peptide sequence.

In one embodiment the peptide analogue comprises 2 branched amino acidprobes, wherein one branched amino acid probe is covalently bound to theC-terminus of the peptide and another branched amino acid probe isattached to the side chain amino group of an amino-alkyl amino acidresidue within said peptide sequence.

In one embodiment the peptide analogue comprises 2 branched amino acidprobes, wherein each of the two branched amino acid probes are attachedto the side chain amino group of different (or separate) amino-alkylamino acid residues within said peptide sequence.

In one embodiment of the invention the peptide analogue of the inventioncomprises 3 branched amino acid probes.

In one embodiment the peptide analogue of the invention comprises 3branched amino acid probes, wherein the first branched amino acid probeis covalently bound to the N-terminus of the peptide, the secondbranched amino acid probe is covalently bound to the C-terminus of thepeptide and the third branched amino acid probe is attached to the sidechain amino group of an amino-alkyl amino acid residue within saidpeptide sequence.

In one embodiment the peptide analogue of the invention comprises 3branched amino acid probes, wherein the first branched amino acid probeis covalently bound to the N-terminus of the peptide, and the second andthird branched amino acid probes are each attached to the side chainamino group of different amino-alkyl amino acid residues within saidpeptide sequence.

In one embodiment the peptide analogue of the invention comprises 3branched amino acid probes, wherein the first branched amino acid probeis covalently bound to the C-terminus of the peptide, and the second andthird branched amino acid probes are each attached to the side chainamino group of different amino-alkyl amino acid residues within saidpeptide sequence.

In one embodiment the peptide analogue of the invention comprises 3branched amino acid probes, wherein each of the first, the second andthe third branched amino acid probes are attached to the side chainamino group of different amino-alkyl amino acid residues within saidpeptide sequence.

Peptide Part of the Analogue

The peptide analogue according to the present invention comprises apeptide and one or more branched amino acid probes.

In one embodiment the peptide of the peptide analogue is any peptide,polypeptide or protein, which peptide in one embodiment is native ornaturally occurring, which peptide in one embodiment is a biologicallyactive variant of a naturally occurring peptide.

In one embodiment said peptide is a fragment of a larger polypeptide orprotein.

In one embodiment said peptide is an N-terminal fragment comprising from1-50 of the most N-terminal amino acids of said protein, such as 1-2,2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-14, 14-16, 16-18,18-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 of the most N-terminalamino acids of said protein.

In one embodiment said peptide is a C-terminal fragment comprising from1-50 of the most C-terminal amino acids of said protein, such as 1-2,2-3, 3-4, 4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-14, 14-16, 16-18,18-20, 20-25, 25-30, 30-35, 35-40, 40-45, 45-50 of the most C-terminalamino acids of said protein.

In one embodiment said peptide is a fragment comprising from 1-50consecutive amino acid residues of said protein, such as 1-2, 2-3, 3-4,4-5, 5-6, 6-7, 7-8, 8-9, 9-10, 10-12, 12-14, 14-16, 16-18, 18-20, 20-25,25-30, 30-35, 35-40, 40-45, 45-50 consecutive amino acid residues ofsaid protein.

In some embodiments, the peptide analogues provided herein have one ormore improved properties compared to the native peptide.

A peptide is a single linear polymer chain derived from the condensationof amino acids. Peptides may be distinguished from proteins on the basisof size, and as an arbitrary benchmark can be understood to containapproximately 75 (such as 50) or fewer amino acid residues.

In one embodiment the peptide part of the peptide analogue of thepresent invention is a peptide having (comprising or consisting of) 2 to100 amino acid residues, such as from 2 to 3, for example 3 to 4, suchas 4 to 5, for example 5 to 6, such as 6 to 7, for example 7 to 8, suchas 8 to 9, for example 9 to 10, such as 10 to 12, for example 12 to 14,such as 14 to 16, for example 16 to 18, such as 18 to 20, for example 20to 25, such as 25 to 30, for example 30 to 40, such as 40 to 50, forexample 50 to 75, such as 75 to 100 amino acid residues.

In one embodiment the peptide of the present invention is a peptidehaving less than 50 amino acid residues, such as less than 40, forexample less than 30, amino acid residues.

The sequence of amino acid residues in a native peptide is defined bythe sequence of a gene, which is encoded in the genetic code. Ingeneral, the genetic code specifies 20 standard amino acids naturallyincorporated into polypeptides (proteinogenic): Ala, Arg, Asn, Asp, Cys,GIn, GIu, GIy, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Tyr, Thr, Trp,VaI, and 2 which are incorporated into proteins by unique syntheticmechanisms: Sec (selenocysteine, or U) and Pyl (pyrrolysine, O). Theseare all L-stereoisomers.

Aside from the 22 standard or natural amino acids, there are many othernon-naturally occurring amino acids (non-proteinogenic or non-standard).They are either not found in proteins, or are not produced directly andin isolation by standard cellular machinery.

Non-standard amino acids are usually formed through modifications tostandard amino acids, such as post-translational modifications. Examplesof unnatural amino acid residues are Nle (Norleucine), Orn (ornithine,deguanylated Arginine), Nal (beta-2-naphthyl-alanine), D-Nal(beta-2-naphthyl-D-alanine), D-Arg, D-Trp, D-Phe and D-Val.

Any amino acids according to the present invention may be in the L- orD-configuration. If nothing is specified, reference to the L-isomericform is preferably meant.

The term peptide also embraces post-translational modificationsintroduced by chemical or enzyme-catalyzed reactions, as are known inthe art. Such post-translational modifications can be introduced priorto partitioning, if desired. Also, functional equivalents may comprisechemical modifications such as ubiquitination, labeling (e.g., withradionuclides, various enzymes, etc.), pegylation (derivatization withpolyethylene glycol), or by insertion (or substitution by chemicalsynthesis) of amino acids such as ornithine, which do not normally occurin human proteins.

Peptides with N-terminal alkylations and C-terminal esterifications arealso encompassed within the present invention. Functional equivalentsalso comprise glycosylated and covalent or aggregative conjugates formedwith the same molecules, including dimers or unrelated chemicalmoieties. Such functional equivalents are prepared by linkage offunctionalities to groups which are found in a fragment including at anyone or both of the N- and C-termini, by means known in the art.

In some embodiments, the peptides according to the present invention aremodified by acetylation, such as N-terminal acetylation. In someembodiments the peptides according to the present invention are modifiedby C-terminal amidation.

The peptides of the present invention may be any peptide known to theskilled person as having a biological effect. In one embodiment theaddition of a branched amino acid probe to the peptide in question willinfluence said biological effect, such as enhance or improve abiological effect.

A variant of a peptide of the invention is in one embodiment a peptidederived from the native peptide by fragmentation (such as N-terminalfragments, C-terminal fragments, or fragments from within a peptide),deletion, insertion, mutation or substitution of one or more amino acidresidues (such as conservative amino acid substitution or introductionof non-proteinogenic amino acid residues), and/or modulation of thepeptide such as by acetylation, by insertion of an amino acid in theD-configuration, and other modifications known to the skilled person.

In one embodiment the peptide analogue of the invention comprises one ormore branched amino acid probes and a peptide having animmune-modulating effect (an immune-modulating peptide), in oneembodiment having an anti-inflammatory and/or pro-resolving effect.

In one embodiment the peptide analogue of the invention comprises one ormore branched amino acid probes and a peptide having a metabolic effect.

In one embodiment the peptide analogue of the invention comprises one ormore branched amino acid probes and a peptide having a cardiovasculareffect.

In one embodiment the peptide analogue of the invention comprises one ormore branched amino acid probes and a peptide having an organ protectiveand/or tissue protective effect.

In one embodiment the peptide analogue comprises a peptide hormone, aneurotransmitter, a neuropeptide, a lipopeptide, an enzyme, a growthfactor, a metabologen, a transcription factor, a receptor agonist, areceptor antagonist, a ligand, or a carrier protein.

In one embodiment the peptide analogue comprises a peptide selected fromthe group consisting of VIP (Vasoactive Intestinal Peptide; PHM27),PACAP (Pituitary Adenylate Cyclase Activating Peptide), Peptide PHI 27(Peptide Histidine Isoleucine 27), GHRH 1-24 (Growth Hormone ReleasingHormone 1-24), Glucagon, Secretin, glicentin precursor, GIP (gastricinhibitory peptide), prealbumin or transthyretin (TTR), peptide HI-27and growth hormone releasing factor (GHRF or GHRH), incretins,glucagon-like peptide-1 (GLP-1), GLP-1 (7-37), GLP-1 (7-33),glucagon-like peptide-2 (GLP-2) and exendin-4, or variants thereof.

In one embodiment the peptide analogue comprises a peptide selected fromthe group consisting of somatotrophins (such as somatotropin or growthhormone (GH)), Thyrotrophins (such as Thyroid-stimulating hormone (TSH),Corticotropins (such as Adrenocorticotropic hormone (ACTH), andBeta-endorphin), Lactotrophins (such as Prolactin (PRL), Gonadotropins(such as Luteinizing hormone (LH) and Follicle-stimulating hormone(FSH)), Antidiuretic hormone (ADH or vasopressin) Oxytocin, growthhormone-releasing hormone (GHRH), somatostatin, thyrotropin-releasinghormone (TRH), corticotropin-releasing hormone (CRH)Gonadotropin-Releasing Hormone (GnRH), CREB (cAMP responseelement-binding protein), Lactotripeptides, Isoleucine-Proline-Proline(IPP) and Valine-Proline-Proline (VPP), and variants thereof.

In one embodiment the peptide analogue comprises a pancreaticpolypeptide-related peptide including NPY (NeuroPeptide Y), PYY (PeptideYY), APP (Avian Pancreatic Polypeptide) and PPY/PP (PancreaticPolypeptide), or variants thereof.

In one embodiment the peptide analogue comprises an opioid peptide (oropioid polypeptide hormone/opioid neuropeptide), includingProopiomelanocortin (POMC) peptides (including N-Terminal Peptide ofProopiomelanocortin (NPP, or pro-γ-MSH), γ-MSH, Corticotropin(Adrenocorticotropic Hormone, or ACTH), α-Melanotropin, α-MSH,Corticotropin-like Intermediate Peptide (CLIP), β-Lipotropin (β-LPH),Lipotropin Gamma (γ-LPH), β-MSH, β-Endorphin and [Met]Enkephalin);Enkephalin pentapeptides (Met-enkephalin and Leu-enkephalin),Prodynorphin peptides, dynorphins (dynorphin A, dynorphin B,α-neo-endorphin, β-neo-endorphin, and Big dynorphin), endorphins(beta-endorphin, Alpha-endorphin, Gamma-endorphin, α-neo-endorphin andβ-neo-endorphin), Adrenorphin, Amidorphin, Leumorphin, Nociceptin,Opiorphin, and Spinorphin, or variants thereof.

In one embodiment the peptide analogue comprises a neuropeptide, or aneurotransmitter, including kinins, tachykinin neuropeptides (includingsubstance P, kassinin, neurokinin A (NKA), neurokinin B (NKB), eledoisinand physalaemin), Bradykinin, Neuromedins/Bombesin-related peptides(including Neuromedin B (NMB), Neuromedin N, Neuromedin S and NeuromedinU (NmU)); Angiotensin, Bombesin, Calcitonin gene-related peptide (CGRP),α-CGRP, β-CGRP, Carnosine, Cocaine and amphetamine regulated transcript(CART), Delta sleep-inducing peptide (DSIP), FMRFamide,FMRFamide-related peptides (FaRPs), Galanin, Galanin-like peptide(GALP), Gastrin releasing peptide (GRP), Neuropeptide S, Neuropeptide Y,Neurophysins (Neurophysin I and Neurophysin II), Neurotensin, Pancreaticpolypeptide, Pituitary adenylate cyclase activating peptide (PACAP),RVD-Hpa, hemopressin, VGF (VGF nerve growth factor inducible), andVGF-derived peptides (TLQP-21), or variants thereof.

In one embodiment the peptide analogue comprises a Calcitonin peptide,including Calcitonin, Amylin (or Islet Amyloid Polypeptide (IAPP)) andAGG01, or variants thereof.

In one embodiment the peptide analogue comprises a growth factor,including in one embodiment Adrenomedullin (AM), Angiopoietin (Ang),Autocrine motility factor, Bone morphogenetic proteins (BMPs) (BMP1,BMP2, BMP3, BMP4, BMP5, BMP6, BMP7, BMP8a, BMP8b, BMP10, BMP15),Brain-derived neurotrophic factor (BDNF), Epidermal growth factor (EGF),Erythropoietin (EPO), Fibroblast growth factor (FGF), Glial cellline-derived neurotrophic factor (GDNF), Granulocyte colony-stimulatingfactor (G-CSF), Granulocyte macrophage colony-stimulating factor(GM-CSF), Growth differentiation factor-9 (GDF9), Hepatocyte growthfactor (HGF), Hepatoma-derived growth factor (HDGF), Insulin-like growthfactors (IGF), IGF-1, IGF-2, Migration-stimulating factor, Myostatin(GDF-8), neurotrophins, Neurotrophin-3 (NT-3), Neurotrophin-4 (NT-4),Nerve growth factor (NGF), Platelet-derived growth factor (PDGF),Thrombopoietin (TPO), Transforming growth factor alpha (TGF-α),Transforming growth factor beta (TGF-β), Tumor necrosis factor-alpha(TNF-α), Vascular endothelial growth factor (VEGF), Wnt proteins, Wnt1,Wnt2, Wnt2B, Wnt3, Wnt3A, Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A,Wnt8B, Wnt9A, Wnt9B, Wnt10A, Wnt10B, Wnt11, Wnt16, placental growthfactor (PGF) and Foetal Bovine Somatotrophin (FBS), or variants thereof.

In one embodiment the peptide analogue comprises a peptide of theinsulin/IGF/relaxin family, including in one embodiment insulin andinsulin-like growth factors, IGF-1, IGF-2, IGF-binding proteins, IGFBP1,IGFBP2, IGFBP3, IGFBP4, IGFBP5, IGFBP6, IGFBP7, Relaxin family peptidehormones, relaxin-like (RLN) peptides, insulin-like (INSL) peptides,RLN1 (relaxin 1), RLN2 (relaxin 2), RLN3 (relaxin 3), INSL3(insulin-like peptide 3, Leydig cell-specific insulin-like peptide),INSL4 (insulin-like peptide 4, early placenta insulin-like peptide,ELIP), INSL5 (insulin-like peptide 5) and INSL6 (insulin-like peptide6), or variants thereof.

In one embodiment the peptide analogue comprises a peptide hormone,including in one embodiment Gastrin, gastrin-34, gastrin-17, gastrin-14,pentagastrin, thyroid hormone (T4), Thyrotropin-releasing hormone (TRH),vasopressin, protein hormones, glycoprotein hormones, growth hormone(GH), insulin, LH, FSH, Thyroid-stimulating hormone (thyrotropin, TSH),Angiotensin (AGT), Angiotensin I, Angiotensin II, Angiotensin III,Angiotensin IV, Atrial natriuretic peptide (ANP), NT-proBNP, B-typeNatriuretic Peptide (BNP) and Atrial natriuretic peptide (ANP), orvariants thereof.

In one embodiment the peptide analogue comprises an Annexin protein,including in one embodiment annexin A-I (lipocortin I) and annexin A-II(annexin II), or variants thereof, including thereof, some of which areknown in the art.

Agonist

In one embodiment the peptide analogue of the invention comprises anagonist. The term “agonist” in the present context refers to a substanceor peptide as defined herein, capable of binding to, or in someembodiments, capable of binding to at least some extent and/oractivating a receptor, or in some embodiments, activating a receptor toat least some extent.

An agonist may be an agonist of several different types of receptors,and thus capable of binding and/or activating several different types ofreceptors. Said agonist can also be a selective agonist which only bindsand activates one type of receptor. The term “antagonist” in the presentcontext refers to a substance capable of inhibiting the effect of areceptor agonist.

Full agonists bind (have affinity for) and activate a receptor,displaying full efficacy at that receptor. “Partial agonists” in thepresent context are peptides able to bind and activate a given receptor,but having only partial efficacy at the receptor relative to a fullagonist. Partial agonists can act as antagonists when competing with afull agonist for receptor occupancy and producing a net decrease in thereceptor activation compared to the effects or activation observed withthe full agonist alone.

“Selective agonists” in the present context are compounds which areselective and therefore predominantly bind and activate one type ofreceptor.

Peptides according to the present invention are in one embodiment anagonist capable of binding and activating to some extent one or severalreceptors and can have different binding affinities and/or differentreceptor activation efficacy for different receptors, wherein affinityrefers to the number and size of intermolecular forces between a peptideligand and its receptor, and residence time of the ligand at itsreceptor binding site; and receptor activation efficacy refers to theability of the peptide ligand to produce a biological response uponbinding to the target receptor and the quantitative magnitude of thisresponse.

In some embodiments, such differences in affinity and receptoractivation efficacy are determined by receptor binding/activationstudies which are conventional in the art, for instance by generatingEC₅₀ and Emax values for stimulation of ligand binding in cellsexpressing one or several types of receptors, or on tissues expressingthe different types of receptors. High affinity means that a lowerconcentration of a compound is needed to obtain a binding of 50% of thereceptors compared to peptides which have lower affinity; high receptoractivation efficacy means that a lower concentration of the peptide isneeded to obtain a 50% receptor activation response (low EC₅₀ value),compared to peptides which have lower affinity and/or receptor activityefficacy (higher EC₅₀ value).

Melanocortins

In one embodiment the peptide of the peptide analogue of the inventionis a melanocortin.

In one embodiment the melanocortin is selected from α-MSH, γ-MSH(comprising γ1-MSH and γ-MSH) and β-MSH, or variants thereof.

In one embodiment the melanocortin is selected from α-MSH and γ-MSH, orvariants thereof.

In one embodiment, a peptide variant is a biologically active variant ofthe peptide, i.e. a variant which retains at least one function of thenative (non-variant) peptide.

α-MSH (SEQ ID NO: 1) Ac-Ser-Tyr-Ser-Met-Glu- His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO: 1) SYSMEHFRWGKPV P01189[138-150],Pro-opiomelanocortin, Homo sapiens aa modification:Valine amide (pos 150) γ1-MSH (SEQ ID NO: 2) Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly (SEQ ID NO: 2) YVMGHFRWDRFG P01189[77-88],Pro-opiomelanocortin, Homo sapiens aa modifications: Phenylalanine amide(pos 88) γ2-MSH (SEQ ID NO: 3) Tyr-Val-Met-Gly-His- Phe-Arg-Trp-Asp-Arg-Phe (SEQ ID NO: 3) YVMGHFRWDRF P01189[77-87], Pro-opiomelanocortin,Homo sapiens aa modifications: Phenylalanine amide (pos 87)

It is thus an aspect of the present invention to provide a melanocortinanalogue comprising a melanocortin peptide and one or more branchedamino acid probes,

wherein said branched amino acid probe comprises a first amino-alkylamino acid residue,said first amino-alkyl amino acid residue optionally being covalentlylinked to a second amino-alkyl amino acid residue, or to a second and athird amino-alkyl amino acid residue, to form a linear chain of 2 or 3amino-alkyl amino acid residues,wherein the side chain(s) of one or more of said first, second and/orthird amino-alkyl amino acid residues are each modified by attaching tothe side chain amino group a molecule independently selected from thegroup consisting of AAA_(q)-AAA; (aa₃)_(p)-AAA_(q); AAA_(q)-(aa₃)_(p);[(aa₃)-AAA]_(p) and [AAA-(aa₃)]p;wherein q is a number selected from 0, 1, 2 and 3; p is a numberselected from 1, 2 and 3; AAA is an amino-alkyl amino acid residue; and(aa₃) is an amino acid residue independently selected from Arg, His, Glyand Ala,wherein said amino-alkyl amino acid residues are optionally acetylated,wherein said first amino-alkyl amino acid residue is covalently linkedto the N-terminus of said melanocortin peptide, covalently linked to theC-terminus of said melanocortin peptide, and/or attached to the sidechain amino group of an amino-alkyl amino acid residue within saidmelanocortin peptide,with the proviso that said branched amino acid probe consists of 2 to 9amino acid residues.

In one embodiment with the proviso that when the melanocortin analoguecomprises one branched amino acid probe comprising one or more lysineresidues, said branched amino acid probe is not attached to theN-terminus of said melanocortin.

In one embodiment with the proviso that when the melanocortin peptide ofthe analogue is α-MSH or γ-MSH, or variants thereof, said branched aminoacid probe is not attached to the N-terminus of said melanocortin.

In one embodiment with the proviso that when the melanocortin analoguecomprises one branched amino acid probe which comprises one or morelysine residues, and the melanocortin peptide of the analogue is α-MSHor γ-MSH, or variants thereof, said branched amino acid probe is notattached to the N-terminus of said melanocortin.

In one embodiment the first amino-alkyl amino acid residue(s) of the oneor more branched amino acid probes is not covalently linked to theN-terminus of said melanocortin peptide.

In one embodiment the first amino-alkyl amino acid residue(s) of the oneor more branched amino acid probes is not covalently linked to theN-terminus of said melanocortin peptide, provided i) said melanocortinanalogues comprise one (1) branched amino acid probe, ii) said branchedamino acid probe comprises one or more lysine residues, and/or iii) saidmelanocortin analogues comprise a melanocortin peptide selected from thegroup consisting of α-MSH and γ-MSH (comprising γ1-MSH and γ-MSH), orvariants thereof.

In one embodiment said melanocortin analogue comprise one branched aminoacid probe, which probe is covalently linked to the C-terminus of saidpeptide, or attached to the side chain amino group of an amino-alkylamino acid residue within said melanocortin peptide.

In one embodiment the amino-alkyl amino acid residue within saidmelanocortin peptide is a lysine residue.

In one embodiment the amino-alkyl amino acid residue within saidmelanocortin peptide is an ornithine residue.

In one embodiment said melanocortin analogue is an α-MSH analogue andcomprise a branched amino acid probe attached to the ε-amino group ofthe lysine residue (Lys) comprised in the native α-MSH peptide(underlined: Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val)(SEQ ID NO: 1).

In one embodiment of the invention the melanocortin peptide analoguecomprises 2 branched amino acid probes, wherein i) one branched aminoacid probe is covalently bound to the N-terminus and another branchedamino acid probe is covalently bound to the C-terminus of themelanocortin peptide; ii) one branched amino acid probe is covalentlybound to the N-terminus and another branched amino acid probe isattached to the side chain amino group of an amino-alkyl amino acidresidue within said melanocortin peptide; iii) one branched amino acidprobe is covalently bound to the C-terminus and another branched aminoacid probe is attached to the side chain amino group of an amino-alkylamino acid residue within said melanocortin peptide; or iv) each of thetwo branched amino acid probes are attached to the side chain aminogroups of different (or separate) amino-alkyl amino acid residues withinsaid melanocortin peptide.

In one embodiment of the invention the peptide analogue of the inventioncomprises 3 branched amino acid probes, wherein each of the first, thesecond and the third branched amino acid probes are covalently bound tothe N-terminus of the peptide, covalently bound to the C-terminus of thepeptide or attached to the side chain amino group of an amino-alkylamino acid residue within said melanocortin peptide.

In one embodiment the melanocortin analogue of the present inventioncomprises an α-MSH peptide, or variants thereof.

In one embodiment the melanocortin analogue of the present inventioncomprises a γ-MSH peptide, or variants thereof.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and a melanocortinpeptide having the amino acid sequence:

(aa₁)_(n)-Y-(aa₂)_(m)-Z

wherein Y is an amino acid sequence consisting of 4 contiguous aminoacid residues selected from the group consisting of His-Phe-Arg-Trp (SEQID NO: 4); His-(D-Phe)-Arg-Trp (SEQ ID NO: 5); His-Phe-(D-Arg)-Trp (SEQID NO: 6); His-Phe-Arg-(D-Trp) (SEQ ID NO: 7); His-(D-Phe)-Arg-(D-Trp)(SEQ ID NO: 8); His-Nal-Arg-Trp (SEQ ID NO: 9) and His-(D-Nal)-Arg-Trp(SEQ ID NO: 10); andwherein Z is an amino acid sequence consisting of 2 or 3 contiguousamino acid residues selected from the group consisting of Lys-Pro-Val;Lys-Pro-(D-Val); Arg-Phe-Gly; Arg-(D-Phe)-Gly; Arg-Phe and Arg-(D-Phe);andwherein n is a number selected from 0, 1, 2, 3, 4 and 5, and (aa₁)independently can be any natural or unnatural amino acid residue, andwherein m is 0 or 1, and (aa₂) can be any one natural or unnatural aminoacid residue.

In one embodiment said melanocortin peptide is α-MSH or γ-MSH or isderived from α-MSH or γ-MSH.

In one embodiment (aa₁)_(n) is a sequence consisting of from 0 to 5amino acids (n=0, 1, 2, 3, 4, or 5). In a particular embodiment,(aa₁)_(n) is a sequence consisting of 4 or 5 contiguous amino acids (n=4or 5).

In one embodiment, (aa₁)_(n) is selected from the group consisting ofSer-Tyr-Ser-Met-Glu (SEQ ID NO: 11), Ser-Tyr-Ser-Nle-Glu (SEQ ID NO: 12)and Ser-Ser-Ile-Ile-Ser (SEQ ID NO: 13), wherein said N-terminal Ser isoptionally acetylated.

In one embodiment, (aa₁)_(n) is selected from the group consisting ofSer-Tyr-Ser-Met-Glu (SEQ ID NO: 11), Ser-Tyr-Ser-Nle-Glu (SEQ ID NO:12), Ser-Ser-Ile-Ile-Ser (SEQ ID NO: 13), Ac-Ser-Tyr-Ser-Met-Glu (SEQ IDNO: 11), Ac-Ser-Tyr-Ser-Nle-Glu (SEQ ID NO: 12) andAc-Ser-Ser-Ile-Ile-Ser (SEQ ID NO: 13).

In one embodiment, (aa₁)_(n) is selected from the group consisting ofTyr-Val-Met-Gly (SEQ ID NO: 14) and Tyr-Val-Nle-Gly (SEQ ID NO: 15).

In one embodiment (aa₂)_(m) is selected from the group consisting of Glyand Asp. In one embodiment (aa₂)_(m) is Gly. In another embodiment(aa₂)_(m) is Asp.

In one embodiment, Z is Lys-Pro-Val or Lys-Pro-(D-Val). In anotherembodiment, Z is Arg-Phe-Gly or Arg-(D-Phe)-Gly. In yet anotherembodiment, Z is Arg-Phe or Arg-(D-Phe).

In one embodiment, the most carboxy terminal amino acid of themelanocortin peptide is amidated (—NH₂; —CONH₂). Thus, in oneembodiment, Val or (D-Val) is a Valine amide. In another embodiment, Pheor (D-Phe) is a Phenylalanine amide. In yet another embodiment, Gly isGlycine amide.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and an α-MSH peptidehaving the amino acid sequence:

(aa₁)_(n)-Y-(aa₂)_(m)-Z

wherein n is a number selected from 0, 1, 2, 3, 4 and 5, and (aa₁)independently is any natural or unnatural amino acid residue, andwherein m is 0 or 1, and (aa₂) is any natural or unnatural amino acidresidue,wherein Y is selected from the group consisting of His-Phe-Arg-Trp (SEQID NO: 4); His-(D-Phe)-Arg-Trp (SEQ ID NO: 5); His-Phe-(D-Arg)-Trp (SEQID NO: 6); His-Phe-Arg-(D-Trp) (SEQ ID NO: 7); His-(D-Phe)-Arg-(D-Trp)(SEQ ID NO: 8); His-Nal-Arg-Trp (SEQ ID NO: 9) and His-(D-Nal)-Arg-Trp(SEQ ID NO: 10); andwherein Z is selected from the group consisting of Lys-Pro-Val andLys-Pro-(D-Val), and Val or (D-Val) is optionally valine amide.

In one embodiment (aa₁)_(n) is selected from the group consisting ofSer-Tyr-Ser-Met-Glu (SEQ ID NO: 11), Ser-Tyr-Ser-Nle-Glu (SEQ ID NO:12), Ser-Ser-Ile-Ile-Ser (SEQ ID NO: 13), Ac-Ser-Tyr-Ser-Met-Glu (SEQ IDNO: 11), Ac-Ser-Tyr-Ser-Nle-Glu (SEQ ID NO: 12) andAc-Ser-Ser-Ile-Ile-Ser (SEQ ID NO: 13). In one embodiment (aa₂)_(m) isGly.

In one embodiment the melanocortin peptide is derived from α-MSH and(aa₁)_(n) is Ser-Tyr-Ser-Met-Glu (SEQ ID NO: 11), Ser-Tyr-Ser-Nle-Glu(SEQ ID NO: 12) or Ser-Ser-Ile-Ile-Ser (SEQ ID NO: 13); Y isHis-Phe-Arg-Trp (SEQ ID NO: 4), His-(D-Phe)-Arg-Trp (SEQ ID NO: 5) orHis-Phe-Arg-(D-Trp) (SEQ ID NO: 7); (aa₂)_(m) is Gly and Z isLys-Pro-Val.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and an α-MSH peptideselected from the group consisting of

(SEQ ID NO: 1) Ser-Tyr-Ser-Met-Glu- His-Phe-Arg-Trp-Gly- Lys-Pro-Val,(SEQ ID NO: 16) Ser-Tyr-Ser-Met-Glu- His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val, (SEQ ID NO: 17) Ser-Tyr-Ser-Met-Glu-His-Phe-(D-Arg)-Trp-Gly- Lys-Pro-Val, (SEQ ID NO: 18)Ser-Tyr-Ser-Met-Glu- His-Phe-Arg-(D-Trp)-Gly- Lys-Pro-Val,(SEQ ID NO: 19) Ser-Tyr-Ser-Met-Glu- His-(D-Phe)-Arg-(D-Trp)-Gly-Lys-Pro-Val, (SEQ ID NO: 20) Ser-Tyr-Ser-Met-Glu- His-Nal-Arg-Trp-Gly-Lys-Pro-Val, (SEQ ID NO: 21) Ser-Tyr-Ser-Met-Glu-His-(D-Nal)-Arg-Trp-Gly- Lys-Pro-Val, (SEQ ID NO: 22)Ser-Tyr-Ser-Met-Glu- His-Phe-Arg-Trp-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 23) Ser-Tyr-Ser-Met-Glu- His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val), (SEQ ID NO: 24) Ser-Tyr-Ser-Met-Glu-His-Phe-(D-Arg)-Trp-Gly- Lys-Pro-(D-Val), (SEQ ID NO: 25)Ser-Tyr-Ser-Met-Glu- His-Phe-Arg-(D-Trp)-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 26) Ser-Tyr-Ser-Met-Glu- His-(D-Phe)-Arg-(D-Trp)-Gly-Lys-Pro-(D-Val), (SEQ ID NO: 27) Ser-Tyr-Ser-Met-Glu-His-Nal-Arg-Trp-Gly- Lys-Pro-(D-Val), (SEQ ID NO: 28)Ser-Tyr-Ser-Met-Glu- His-(D-Nal)-Arg-Trp-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 29) Ser-Tyr-Ser-Nle-Glu- His-Phe-Arg-Trp-Gly- Lys-Pro-Val,(SEQ ID NO: 30) Ser-Tyr-Ser-Nle Glu- His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-Val, (SEQ ID NO: 31) Ser-Tyr-Ser-Nle Glu-His-Phe-(D-Arg)-Trp-Gly- Lys-Pro-Val, (SEQ ID NO: 32)Ser-Tyr-Ser-Nle-Glu- His-Phe-Arg-(D-Trp)-Gly- Lys-Pro-Val,(SEQ ID NO: 33) Ser-Tyr-Ser-Nle-Glu- His-(D-Phe)-Arg-(D-Trp)-Gly-Lys-Pro-Val, (SEQ ID NO: 34) Ser-Tyr-Ser-Nle-Glu- His-Nal-Arg-Trp-Gly-Lys-Pro-Val, (SEQ ID NO: 35) Ser-Tyr-Ser-Nle-Glu-His-(D-Nal)-Arg-Trp-Gly- Lys-Pro-Val, (SEQ ID NO: 36)Ser-Tyr-Ser-Nle-Glu- His-Phe-Arg-Trp-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 37) Ser-Tyr-Ser-Nle-Glu- His-(D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val), (SEQ ID NO: 38) Ser-Tyr-Ser-Nle-Glu-His-Phe-(D-Arg)-Trp-Gly- Lys-Pro-(D-Val), (SEQ ID NO: 39)Ser-Tyr-Ser-Nle-Glu- His-Phe-Arg-(D-Trp)-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 40) Ser-Tyr-Ser-Nle-Glu- His-(D-Phe)-Arg-(D-Trp)-Gly-Lys-Pro-(D-Val), (SEQ ID NO: 41) Ser-Tyr-Ser-Nle-Glu-His-Nal-Arg-Trp-Gly- Lys-Pro-(D-Val), (SEQ ID NO: 42)Ser-Tyr-Ser-Nle-Glu- His-(D-Nal)-Arg-Trp-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 43) Ser-Ser-Ile-Ile-Ser-His- Phe-Arg-Trp-Gly- Lys-Pro-Val,(SEQ ID NO: 44) Ser-Ser-Ile-Ile-Ser-His- (D-Phe)-Arg-Trp-Gly-Lys-Pro-Val, (SEQ ID NO: 45) Ser-Ser-Ile-Ile-Ser-His-Phe-(D-Arg)-Trp-Gly- Lys-Pro-Val, (SEQ ID NO: 46)Ser-Ser-Ile-Ile-Ser-His- Phe-Arg-(D-Trp)-Gly- Lys-Pro-Val,(SEQ ID NO: 47) Ser-Ser-Ile-Ile-Ser-His- (D-Phe)-Arg-(D-Trp)-Gly-Lys-Pro-Val, (SEQ ID NO: 48) Ser-Ser-Ile-Ile-Ser-His- Nal-Arg-Trp-Gly-Lys-Pro-Val, (SEQ ID NO: 49) Ser-Ser-Ile-Ile-Ser-His-(D-Nal)-Arg-Trp-Gly- Lys-Pro-Val, (SEQ ID NO: 50)Ser-Ser-Ile-Ile-Ser-His- Phe-Arg-Trp-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 51) Ser-Ser-Ile-Ile-Ser-His- (D-Phe)-Arg-Trp-Gly-Lys-Pro-(D-Val), (SEQ ID NO: 52) Ser-Ser-Ile-Ile-Ser-His-Phe-(D-Arg)-Trp-Gly- Lys-Pro-(D-Val), (SEQ ID NO: 53)Ser-Ser-Ile-Ile-Ser-His- Phe-Arg-(D-Trp)-Gly- Lys-Pro-(D-Val),(SEQ ID NO: 54) Ser-Ser-Ile-Ile-Ser-His- (D-Phe)-Arg-(D-Trp)-Gly-Lys-Pro-(D-Val), (SEQ ID NO: 55) Ser-Ser-Ile-Ile-Ser-His-Nal-Arg-Trp-Gly- Lys-Pro-(D-Val), and (SEQ ID NO: 56)Ser-Ser-Ile-Ile-Ser-His- (D-Nal)-Arg-Trp-Gly- Lys-Pro-(D-Val),wherein the most carboxy terminal Val or (D-Val) is optionally a Valineamide.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and a γ-MSH peptidehaving the amino acid sequence:

(aa₁)_(n)-Y-(aa₂)_(m)-Z

wherein n is a number selected from 0, 1, 2, 3, 4 and 5, and (aa₁)independently is any natural or unnatural amino acid residue, andwherein m is 0 or 1, and (aa₂) is any natural or unnatural amino acidresidue,wherein Y is selected from the group consisting of His-Phe-Arg-Trp (SEQID NO: 4); His-(D-Phe)-Arg-Trp (SEQ ID NO: 5); His-Phe-(D-Arg)-Trp (SEQID NO: 6); His-Phe-Arg-(D-Trp) (SEQ ID NO: 7); His-(D-Phe)-Arg-(D-Trp)(SEQ ID NO: 8); His-Nal-Arg-Trp (SEQ ID NO: 9) and His-(D-Nal)-Arg-Trp(SEQ ID NO: 10); andwherein Z is selected from the group consisting of Arg-Phe-Gly,Arg-(D-Phe)-Gly, Arg-Phe and Arg-(D-Phe); and Phe or (D-Phe) isphenylalanine amide or Gly is glycine amide.

In one embodiment (aa₁)_(n) is selected from the group consisting ofTyr-Val-Met-Gly (SEQ ID NO: 14) and Tyr-Val-Nle-Gly (SEQ ID NO: 15). Inone embodiment (aa₂)_(m) is Asp.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and a γ1-MSH peptideselected from the group consisting of

(SEQ ID NO: 2) Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp- Arg-Phe-Gly,(SEQ ID NO: 57) Tyr-Val-Met-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-Phe-Gly,(SEQ ID NO: 58) Tyr-Val-Met-Gly-His-Phe-(D-Arg)-Trp- Asp-Arg-Phe-Gly,(SEQ ID NO: 59) Tyr-Val-Met-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-Phe-Gly,(SEQ ID NO: 60) Tyr-Val-Met-Gly-His-(D-Phe)-Arg-(D-Trp)-Asp-Arg-Phe-Gly, (SEQ ID NO: 61)Tyr-Val-Met-Gly-His-Nal-Arg-Trp-Asp- Arg-Phe-Gly, (SEQ ID NO: 62)Tyr-Val-Met-Gly-His-(D-Nal)-Arg-Trp- Asp-Arg-Phe-Gly, (SEQ ID NO: 63)Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp- Arg-(D-Phe)-Gly, (SEQ ID NO: 64)Tyr-Val-Met-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-(D-Phe)-Gly,(SEQ ID NO: 65) Tyr-Val-Met-Gly-His-Phe-(D-Arg)-Trp-Asp-Arg-(D-Phe)-Gly, (SEQ ID NO: 66)Tyr-Val-Met-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-(D-Phe)-Gly,(SEQ ID NO: 67) Tyr-Val-Met-Gly-His-(D-Phe)-Arg-(D-Trp)-Asp-Arg-(D-Phe)-Gly, (SEQ ID NO: 68)Tyr-Val-Met-Gly-His-Nal-Arg-Trp-Asp- Arg-(D-Phe)-Gly, (SEQ ID NO: 69)Tyr-Val-Met-Gly-His-(D-Nal)-Arg-Trp- Asp-Arg-(D-Phe)-Gly,(SEQ ID NO: 70) Tyr-Val-Nle-Gly-His-Phe-Arg-Trp-Asp- Arg-Phe-Gly,(SEQ ID NO: 71) Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-Phe-Gly,(SEQ ID NO: 72) Tyr-Val-Nle-Gly-His-Phe-(D-Arg)-Trp- Asp-Arg-Phe-Gly,(SEQ ID NO: 73) Tyr-Val-Nle-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-Phe-Gly,(SEQ ID NO: 74) Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-(D-Trp)-Asp-Arg-Phe-Gly, (SEQ ID NO: 75)Tyr-Val-Nle-Gly-His-Nal-Arg-Trp-Asp- Arg-Phe-Gly, (SEQ ID NO: 76)Tyr-Val-Nle-Gly-His-(D-Nal)-Arg-Trp- Asp-Arg-Phe-Gly, (SEQ ID NO: 77)Tyr-Val-Nle-Gly-His-Phe-Arg-Trp-Asp- Arg-(D-Phe)-Gly, (SEQ ID NO: 78)Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-(D-Phe)-Gly,(SEQ ID NO: 79) Tyr-Val-Nle-Gly-His-Phe-(D-Arg)-Trp-Asp-Arg-(D-Phe)-Gly, (SEQ ID NO: 80)Tyr-Val-Nle-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-(D-Phe)-Gly,(SEQ ID NO: 81) Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-(D-Trp)-Asp-Arg-(D-Phe)-Gly, (SEQ ID NO: 82)Tyr-Val-Nle-Gly-His-Nal-Arg-Trp-Asp- Arg-(D-Phe)-Gly, and(SEQ ID NO: 83) Tyr-Val-Nle-Gly-His-(D-Nal)-Arg-Trp-Asp-Arg-(D-Phe)-Gly,wherein the most carboxy terminal Gly is optionally glycine amide.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and a γ2-MSH peptideselected from the group consisting of

(SEQ ID NO: 3) Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp- Arg-Phe,(SEQ ID NO: 84) Tyr-Val-Met-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-Phe,(SEQ ID NO: 85) Tyr-Val-Met-Gly-His-Phe-(D-Arg)-Trp- Asp-Arg-Phe,(SEQ ID NO: 86) Tyr-Val-Met-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-Phe,(SEQ ID NO: 87) Tyr-Val-Met-Gly-His-(D-Phe)-Arg-(D- Trp)-Asp-Arg-Phe,(SEQ ID NO: 88) Tyr-Val-Met-Gly-His-Nal-Arg-Trp-Asp- Arg-Phe,(SEQ ID NO: 89) Tyr-Val-Met-Gly-His-(D-Nal)-Arg-Trp- Asp-Arg-Phe,(SEQ ID NO: 90) Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp- Arg-(D-Phe),(SEQ ID NO: 91) Tyr-Val-Met-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-(D-Phe),(SEQ ID NO: 92) Tyr-Val-Met-Gly-His-Phe-(D-Arg)-Trp- Asp-Arg-(D-Phe),(SEQ ID NO: 93) Tyr-Val-Met-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-(D-Phe),(SEQ ID NO: 94) Tyr-Val-Met-Gly-His-(D-Phe)-Arg-(D-Trp)-Asp-Arg-(D-Phe), (SEQ ID NO: 95)Tyr-Val-Met-Gly-His-Nal-Arg-Trp-Asp- Arg-(D-Phe), (SEQ ID NO: 96)Tyr-Val-Met-Gly-His-(D-Nal)-Arg-Trp- Asp-Arg-(D-Phe), (SEQ ID NO: 97)Tyr-Val-Nle-Gly-His-Phe-Arg-Trp-Asp- Arg-Phe, (SEQ ID NO: 98)Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-Phe, (SEQ ID NO: 99)Tyr-Val-Nle-Gly-His-Phe-(D-Arg)-Trp- Asp-Arg-Phe, (SEQ ID NO: 100)Tyr-Val-Nle-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-Phe, (SEQ ID NO: 101)Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-(D- Trp)-Asp-Arg-Phe, (SEQ ID NO: 102)Tyr-Val-Nle-Gly-His-Nal-Arg-Trp-Asp- Arg-Phe, (SEQ ID NO: 103)Tyr-Val-Nle-Gly-His-(D-Nal)-Arg-Trp- Asp-Arg-Phe, (SEQ ID NO: 104)Tyr-Val-Nle-Gly-His-Phe-Arg-Trp-Asp- Arg-(D-Phe), (SEQ ID NO: 105)Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-Trp- Asp-Arg-(D-Phe), (SEQ ID NO: 106)Tyr-Val-Nle-Gly-His-Phe-(D-Arg)-Trp- Asp-Arg-(D-Phe), (SEQ ID NO: 107)Tyr-Val-Nle-Gly-His-Phe-Arg-(D-Trp)- Asp-Arg-(D-Phe), (SEQ ID NO: 108)Tyr-Val-Nle-Gly-His-(D-Phe)-Arg-(D- Trp)-Asp-Arg-(D-Phe),(SEQ ID NO: 109) Tyr-Val-Nle-Gly-His-Nal-Arg-Trp-Asp- Arg-(D-Phe), and(SEQ ID NO: 110) Tyr-Val-Nle-Gly-His-(D-Nal)-Arg-Trp- Asp-Arg-(D-Phe),wherein the most carboxy terminal Phe or (D-Phe) is optionally aPhenylalanine amide.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and a cyclic lactamanalogue of α-MSH, in one embodiment a side-chain cyclic lactam analogueof α-MSH.

In one embodiment the cyclic lactam analogue of α-MSH is selected fromthe group consisting of Ac-Nle-cyclo[Asp-His-D-Phe-Arg-Trp-Lys]-NH₂ (SEQID NO: 111) (MT-II, Melanotan II); SHU9005, SHU9119, [DNal(1′)]-MTII,[Nal(2′)]-MTII, a cyclic α-MSH(1-13) lactam analog; a side-chain cycliclactam analogue of a fragment of α-MSH; a cyclic α-MSH(4-10) lactamanalogue; MBX36; MBX37; Ac-Nle4-cyclo[Asp5, D-Phe7,Lys10]α-MSH-(4-10)-NH2; cyclic disulphide α-MSH (4-10) analogue; a cyclicα-MSH(4-11) lactam analogue; Ac-[Nle4,D-Orn5,Glu8]alpha-MSH4-11-NH;Ac-[Nle4,D-Orn5,D-Phe7,Glu8]alpha-MSH4-11-NH2;[N-Acetyl-Cys4,D-Phe7,Cys10]-α-MSH (4-13), cyclic; or variants thereof.

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and a metal-cyclizedα-MSH analogue; in one embodiment1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) coupledReO-cyclized [Cys3,4,10,-D-Phe7]-MSH3-13 (DOTA-ReCCMSH).

In one embodiment the melanocortin analogue of the present inventioncomprises one or more branched amino acid probes and a disulfide bridgeproviding a cyclized derivative of α-MSH, such as in one embodimentMBJ-06 (WO1998027113).

Disclaimer

In one particular embodiment, the peptide analogue of the presentinvention does not comprise an α-MSH and γ-MSH analogue as disclosed inPCT/EP2013/071935.

In one embodiment, the present peptide analogue does not comprise anα-MSH and/or a γ-MSH peptide, or variants thereof

In one embodiment, the present peptide analogue comprises a peptideother than an α-MSH and/or a γ-MSH peptide, or variants thereof.

In one embodiment the peptide analogue does not comprise an α-MSH and/orγ-MSH peptide having a branched amino acid probe covalently linked (orlinked by a peptide bond) to the most N-terminal amino acid of saidα-MSH and/or γ-MSH peptide.

In one embodiment the peptide analogue does not comprise an α-MSH and/orγ-MSH peptide having a branched amino acid probe, which probe comprisesat least one lysine residue, wherein said branched amino acid probe iscovalently linked to the most N-terminal amino acid of said α-MSH and/orγ-MSH peptide.

The most N-terminal amino acid of α-MSH is Ser (underlined:Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val (SEQ ID NO: 1).

The most N-terminal amino acid of γ-MSH is Tyr (underlined:Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe(-Gly) (SEQ ID NO: 2)

In one embodiment wherein the peptide analogue of the inventioncomprises one (1) branched amino acid probe, wherein one or more (or atleast one) of the amino-alkyl amino acid residues of the branched aminoacid probe is lysine, and wherein the peptide is α-MSH and/or γ-MSH, orvariants thereof, said branched amino acid probe is not attached to theN-terminus of said peptide.

In one embodiment the first amino-alkyl amino acid residue(s) of the oneor more branched amino acid probes is not covalently linked to theN-terminus of said peptide, provided i) said peptide analogues compriseone (1) branched amino acid probe, ii) said branched amino acid probecomprises one or more lysine residues, and/or iii) said peptide analoguecomprise a melanocortin peptide selected from the group consisting ofα-MSH and γ-MSH (comprising γ1-MSH and γ-MSH).

In one embodiment the peptide analogue of the invention does notcomprise or include: A peptide consisting of from 8 to 22 amino acidresidues comprising the amino acid sequence: X-(aa₁)_(n)-Y-(aa₂)_(m)-Z

wherein X comprises a branched amino acid probe having a first lysineresidue (Lys₁) selected from Lys and D-Lys, said first lysine residuebeing linked by a peptide bond to (aa₁)_(n),said first lysine residue being optionally linked by peptide bonds to asecond lysine residue (Lys₂), or to a second and third lysine residue(Lys₃), to form a linear chain of a total of 2 or 3 lysine residuesselected from Lys and D-Lys,wherein the side chain(s) of one or more of each of said first, secondand/or third lysine residues are modified by attaching to the ε-aminogroup of said one or more of each of said lysine residues a moleculeindependently selected from the group consisting of Lys_(q)-Lys;(aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p) and[Lys-(aa₃)]_(p), wherein q is a number selected from 0, 1, 2 and 3; p isa number selected from 1, 2 and 3, and (aa₃) is an amino acid residueindependently selected from Arg, His, Gly and Ala,with the proviso that X consists of from 2 to 9 amino acid residues,wherein Y comprises an amino acid sequence consisting of 4 contiguousamino acid residues selected from the group consisting ofHis-Phe-Arg-Trp (SEQ ID NO: 4); His-(D-Phe)-Arg-Trp (SEQ ID NO: 5);His-Phe-(D-Arg)-Trp (SEQ ID NO: 6); His-Phe-Arg-(D-Trp) (SEQ ID NO: 7);His-(D-Phe)-Arg-(D-Trp) (SEQ ID NO: 8); His-Nal-Arg-Trp (SEQ ID NO: 9)and His-(D-Nal)-Arg-Trp (SEQ ID NO: 10); andwherein Z comprises an amino acid sequence consisting of 2 or 3contiguous amino acid residues selected from the group consisting ofLys-Pro-Val; Lys-Pro-(D-Val); Arg-Phe-Gly; Arg-(D-Phe)-Gly; Arg-Phe andArg-(D-Phe); andwherein n is a number selected from 0, 1, 2, 3, 4 and 5, and (aa₁)independently can be any natural or unnatural amino acid residue, andwherein m is 0 or 1, and (aa₂) can be any natural or unnatural aminoacid residue.

In one embodiment the peptide analogue of the invention does notcomprise or include:

A peptide consisting of from 8 to 22 amino acid residues comprising theamino acid sequence: X-(aa₁)_(n)-Y-(aa₂)_(m)-Z

wherein X comprises a branched amino acid probe having a first lysineresidue (Lys₁) selected from Lys and D-Lys, said first lysine residuebeing linked by a peptide bond to (aa₁)_(n),said first lysine residue being optionally linked by peptide bonds to asecond lysine residue (Lys₂), or to a second and third lysine residue(Lys₃), to form a linear chain of a total of 2 or 3 lysine residuesselected from Lys and D-Lys,wherein the side chain(s) of one or more of each of said first, secondand/or third lysine residues are modified by attaching to the ε-aminogroup of said one or more of each of said lysine residues a moleculeindependently selected from the group consisting of Lys_(q)-Lys;(aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p); [(aa₃)-Lys]_(p) and[Lys-(aa₃)]_(p), wherein q is a number selected from 0, 1, 2 and 3; p isa number selected from 1, 2 and 3, and (aa₃) is an amino acid residueindependently selected from Arg, His, Gly and Ala,with the proviso that X consists of from 2 to 9 amino acid residues,wherein Y comprises an amino acid sequence consisting of 4 contiguousamino acid residues selected from the group consisting ofHis-Phe-Arg-Trp (SEQ ID NO: 4); His-(D-Phe)-Arg-Trp (SEQ ID NO: 5);His-Phe-(D-Arg)-Trp (SEQ ID NO: 6); His-Phe-Arg-(D-Trp) (SEQ ID NO: 7);His-(D-Phe)-Arg-(D-Trp) (SEQ ID NO: 8); His-Nal-Arg-Trp (SEQ ID NO: 9)and His-(D-Nal)-Arg-Trp (SEQ ID NO: 10); andwherein Z comprises an amino acid sequence consisting of 2 or 3contiguous amino acid residues selected from the group consisting ofLys-Pro-Val; Lys-Pro-(D-Val); Arg-Phe-Gly; Arg-(D-Phe)-Gly; Arg-Phe andArg-(D-Phe); andwherein (aa₁)_(n) is selected from the group consisting ofSer-Tyr-Ser-Met-Glu (SEQ ID NO: 11), Ser-Tyr-Ser-Nle-Glu (SEQ ID NO:12), Ser-Ser-Ile-Ile-Ser (SEQ ID NO: 13), Tyr-Val-Met-Gly (SEQ ID NO:14) and Tyr-Val-Nle-Gly (SEQ ID NO: 15); andwherein (aa₂)_(m) is selected from the group consisting of Gly and Asp.

Methods of Preparation

The peptide analogues according to the present invention may be preparedby any suitable methods known in the art. Thus, in some embodiments thepeptide and the branched amino acid probe are each prepared by standardpeptide-preparation techniques, such as solution synthesis or solidphase peptide synthesis (SPPS) such as Merrifield-type solid phasesynthesis.

The peptide analogues of the invention are in one embodiment prepared bysolid phase synthesis by first constructing the pharmacologically activepeptide sequence, using well-known standard protection, coupling andde-protection procedures, thereafter sequentially coupling the branchedamino acid probe onto the active peptide in a manner similar to theconstruction of the active peptide, and finally cleaving off the entirepeptide analogue from the carrier. This strategy yields a peptide,wherein the branched amino acid probe is covalently bound to thepharmacologically active peptide at the N-terminal nitrogen atom of thepeptide.

In one embodiment, the alpha nitrogen on the final amino acid in thebranched amino acid sequence is capped with acetyl, using standardacylation techniques, prior to or after coupling of the branched aminoacid sequence on the active peptide.

Reactive moieties at the N- and C-termini, which facilitates amino acidcoupling during synthesis, as well as reactive side chain functionalgroups, can interact with free termini or other side chain groups duringsynthesis and peptide elongation and negatively influence yield andpurity. Chemical groups are thus developed that bind to specific aminoacid functional groups and block, or protect, the functional group fromnonspecific reactions. Purified, individual amino acids are reacted withthese protecting groups prior to synthesis and then selectively removedduring specific steps of peptide synthesis. Examples of N-terminalprotecting groups are t-Boc and Fmoc, commonly used in solid-phasepeptide synthesis. C-terminal protecting groups are mostly used inliquid-phase synthesis. Because N-terminal deprotection occurscontinuously during peptide synthesis, protecting schemes have beenestablished in which the different types of side chain protecting groups(benzyl; Bzl or tert-butyl; tBu) are matched to either Boc or Fmoc,respectively, for optimized deprotection.

In a particular embodiment of the invention, when preparing the branchedamino acid probe, exemplified by Ac(Ac-Lys-Lys)Lys-, the protectiongroup for Lys is Mtt, which protected amino acid is commerciallyavailable (Fmoc-Lys(Mtt)-OH; N-α-Fmoc-N-ε-4-methyltrityl-L-lysine, CAS#167393-62-6). Lys(Mtt) allows for capping Lys with acetyl as it is notcleaved under the conditions that cleave Fmoc, and may be removedwithout cleavage of other side chain protection groups.

The method of preparation is in some embodiments optimized by routinemethods in the art that may increase the yield and/or quality of thethus prepared synthetic peptide. For instance, use of pseudoproline(oxazolidine) dipeptides in the Fmoc SPPS of serine- andthreonine-containing peptides may lead to improvements in quality andyield of crude products and may help avoid unnecessary repeat synthesisof failed sequences. These dipeptides are easy to use: simply substitutea serine or threonine residue together with the preceding amino acidresidue in the peptide sequence with the appropriate pseudoprolinedipeptide. The native sequence is regenerated on cleavage anddeprotection.

In one embodiment the sequence of the pharmacologically active peptideand the branched amino acid probe (or parts thereof) are each preparedseparately by for example solution synthesis, solid phase synthesis,recombinant techniques, or enzymatic synthesis, followed by coupling ofthe (at least) two sequences by well-known segment condensationprocedures, either in solution or using solid phase techniques, or acombination thereof.

In one embodiment, the peptides are prepared by recombinant DNA methodsand the branched amino acid probe is prepared by solid or solution phasesynthesis. The conjugation of the peptide and the branched amino acidprobe is in one embodiment carried out by using chemical ligation. Thistechnique allows for the assembling of totally unprotected peptidesegments in a highly specific manner. In another embodiment, theconjugation is performed by protease-catalysed peptide bond formation,which offers a highly specific technique to combine totally unprotectedpeptide segments via a peptide bond.

In one embodiment, the C-terminal amino acid of the branched amino acidprobe or the C-terminal amino acid of the peptide is attached to thesolid support material by means of a common linker such as2,4-dimethoxy-4′-hydroxy-benzophenone,4-(4-hydroxy-methyl-3-methoxyphenoxy)-butyric acid,4-hydroxy-methylbenzoic acid, 4-hydroxymethyl-phenoxyacetic acid,3-(4-hydroxymethylphenoxy)propionic acid, orp-{(R,S)-α-[1-(9H-Fluoren-9-yl)-methoxyformamido]-2,4-dimethoxybenzyl}-phenoxyaceticacid (Rink amide linker).

Examples of suitable solid support materials (SSM) are e.g.,functionalised resins such as polystyrene, polyacrylamide,polydimethylacrylamide, polyethyleneglycol, cellulose, polyethylene,polyethyleneglycol grafted on polystyrene, latex, dynabeads, etc.

The produced peptide analogues of the invention are in some embodimentcleaved from the solid support material by means of an acid such astrifluoracetic acid, trifluoromethanesulfonic acid, hydrogen bromide,hydrogen chloride, hydrogen fluoride, etc. optionally in combinationwith one phenol, thioanisole, etc., or the peptide conjugate of theinvention are in other embodiments cleaved from the solid support bymeans of a base such as ammonia, hydrazine, an alkoxide, such as sodiumethoxide, an hydroxide, such as sodium hydroxide, etc.

In other embodiments, the peptide analogues of the invention may beprepared or produced by recombinant techniques. Thus, in one aspect ofthe present invention the peptide is produced by host cells comprising afirst nucleic acid sequence encoding the peptide or peptide analogueoperably associated with a second nucleic acid capable of directingexpression in said host cells. In some embodiments the second nucleicacid sequence comprises or even consists of a promoter that will directthe expression of protein of interest in said cells. A skilled personwill be readily capable of identifying useful second nucleic acidsequences (e.g. vectors and plasmids) for use in a given host cell.

The process of producing a recombinant peptide in general comprises thesteps of: providing a host cell, preparing a gene expression constructcomprising a first nucleic acid encoding the peptide operably linked toa second nucleic acid capable of directing expression of said protein ofinterest in the host cell, transforming the host cell with the constructand cultivating the host cell, thereby obtaining expression of thepeptide. In one embodiment of the invention, the recombinantly producedpeptide is excreted by the host cells. The host cell include anysuitable host cell known in the art, including prokaryotic cells, yeastcells, insect cells and mammalian cells.

In one embodiment, the recombinant peptide thus produced is isolated byany conventional method and may be linked via conventional peptide bondforming chemistry to any suitably protected branched amino peptidemoiety. The skilled person will be able to identify suitable proteinisolation steps for purifying the peptide.

Methods of Treatment

It is an aspect to provide peptide analogues as defined according to thepresent invention for use as a medicament.

In another aspect, the present invention provides methods for treatment,prevention or alleviation of a medical condition. Such methods accordingto the present invention in one embodiment comprise one or more steps ofadministration or release of an effective amount of a peptide analogueaccording to the present invention, or a pharmaceutical compositioncomprising one or more such peptides, to an individual in need thereof.In one embodiment, such steps of administration or release according tothe present invention are simultaneous, sequential or separate.

An individual in need as referred to herein, is in one embodiment anindividual that benefits from the administration of a peptide orpharmaceutical composition according to the present invention. Such anindividual in one embodiment suffers from a disease or condition or isat risk of suffering therefrom. The individual is in one embodiment anyhuman being, male or female, infant, middle-aged or old. The disorder tobe treated or prevented in the individual in one embodiment relates tothe age of the individual, the general health of the individual, themedications used for treating the individual and whether or not theindividual has a prior history of suffering from diseases or disordersthat may have or have induced the condition in the individual.

The terms “treatment” and “treating” as used herein refer to themanagement and care of a patient for the purpose of combating acondition, disease or disorder. The term is intended to include the fullspectrum of treatments for a given condition from which the patient issuffering, such as administration of the peptide analogue for thepurpose of: alleviating or relieving symptoms or complications; delayingthe progression of the condition, partially arresting the clinicalmanifestations, disease or disorder; curing or eliminating thecondition, disease or disorder; and/or preventing or reducing the riskof acquiring the condition, disease or disorder, wherein “preventing” or“prevention” is to be understood to refer to the management and care ofa patient for the purpose of hindering the development of the condition,disease or disorder, and includes the administration of the activecompounds to prevent or reduce the risk of the onset of symptoms orcomplications. The patient to be treated is preferably a mammal, inparticular a human being. Treatment of animals, such as mice, rats,dogs, cats, cows, horses, sheep and pigs, is, however, also within thescope of the present invention. The patients to be treated according tothe present invention can be of various ages, for example, adults,children, children under 16, children age 6-16, children age 2-16,children age 2 months to 6 years or children age 2 months to 5 years.

The invention is in one embodiment directed to a peptide analogueaccording to the present invention for use in the treatment of anischemic condition, an inflammatory condition and/or a metaboliccondition.

The invention is in one embodiment directed to a method for treatment ofan ischemic condition, an inflammatory condition and/or a metaboliccondition, said method comprising administering an effective amount of apeptide analogue according to the present invention to an individual inneed thereof.

Further Active Ingredients

In some embodiments, the peptide analogues of the present invention arecombined with or comprise one or more further active ingredients whichare understood as other therapeutical compounds or pharmaceuticallyacceptable derivatives thereof.

Methods for treatment according to the present invention in oneembodiment thus further comprise one or more steps of administration ofone or more further active ingredients, either concomitantly orsequentially, and in any suitable ratios.

Methods of treatment according to the present invention in oneembodiment include a step wherein the pharmaceutical composition orpeptide analogue as defined herein is administered simultaneously,sequentially or separately in combination with one or more furtheractive ingredients.

Administration and Dosage

According to the present invention, a composition comprising a peptideanalogue as defined herein is in one embodiment administered toindividuals in need thereof in pharmaceutically effective doses or atherapeutically effective amount.

A therapeutically effective amount of a peptide according to the presentinvention is in one embodiment an amount sufficient to cure, prevent,reduce the risk of, alleviate or partially arrest the clinicalmanifestations of a given disease or disorder and its complications. Theamount that is effective for a particular therapeutic purpose willdepend on the severity and the sort of the disorder as well as on theweight and general state of the subject. An amount adequate toaccomplish this is defined as a “therapeutically effective amount”.

In one embodiment of the present invention, the composition isadministered in doses of from 1 μg/day to 100 mg/day; such as from 1μg/day to 10 μg/day, such as 10 μg/day to 100 μg/day, such as 100 μg/dayto 250 μg/day, such as 250 μg/day to 500 μg/day, such as 500 μg/day to750 μg/day, such as 750 μg/day to 1 mg/day, such as 1 mg/day to 2mg/day, such as 2 mg/day to 5 mg/day, or such as 5 mg/day to 10 mg/day,such as 10 mg/day to 20 mg/day, such as 20 mg/day to 30 mg/day, such as30 mg/day to 40 mg/day, such as 40 mg/day to 50 mg/day, such as 50mg/day to 75 mg/day, or such as 75 mg/day to 100 mg/day.

In one embodiment of the present invention, one single dose of thecomposition is administered and may comprise of from 1 μg/kg body weightto 100 mg/kg body weight; such as from 1 to 10 μg/kg body weight, suchas 10 to 100 μg/day, such as 100 to 250 μg/kg body weight, such as 250to 500 μg/kg body weight, such as 500 to 750 μg/kg body weight, such as750 μg/kg body weight to 1 mg/kg body weight, such as 1 mg/kg bodyweight to 2 mg/kg body weight, such as 2 to 5 mg/kg body weight, such as5 to 10 mg/kg body weight, such as 10 to 20 mg/kg body weight, such as20 to 30 mg/kg body weight, such as 30 to 40 mg/kg body weight, such as40 to 50 mg/kg body weight, such as 50 to 75 mg/kg body weight, or suchas 75 to 100 mg/kg body weight.

In one embodiment, a dose according to the present invention isadministered one or several times per day, such as from 1 to 6 times perday, such as from 1 to 5 times per day, such as from 1 to 4 times perday, such as from 1 to 3 times per day, such as from 1 to 2 times perday, such as from 2 to 4 times per day, such as from 2 to 3 times perday.

Routes of Administration

It will be appreciated that the preferred route of administration willdepend on the general condition and age of the subject to be treated,the nature of the condition to be treated, the location of the tissue tobe treated in the body and the active ingredient chosen.

Systemic Treatment

In one embodiment, the route of administration allows for introducingthe peptide analogue into the blood stream to ultimately target thesites of desired action.

In one embodiment the routes of administration is any suitable routes,such as an enteral route (including the oral, rectal, nasal, pulmonary,buccal, sublingual, transdermal, intracisternal and intraperitonealadministration), and/or a parenteral route (including subcutaneous,intramuscular, intrathecal, intravenous and intradermal administration).

Appropriate dosage forms for such administration may be prepared byconventional techniques.

Parenteral Administration

Parenteral administration is any administration route not being theoral/enteral route whereby the medicament avoids first-pass degradationin the liver. Accordingly, parenteral administration includes anyinjections and infusions, for example bolus injection or continuousinfusion, such as intravenous administration, intramuscularadministration or subcutaneous administration. Furthermore, parenteraladministration includes inhalations and topical administration.

Accordingly, the peptide analogue or composition is in one embodimentadministered topically to cross any mucosal membrane of an animal towhich the substance or peptide is to be given, e.g. in the nose, vagina,eye, mouth, genital tract, lungs, gastrointestinal tract, or rectum, forexample the mucosa of the nose, or mouth, and accordingly, parenteraladministration may also include buccal, sublingual, nasal, rectal,vaginal and intraperitoneal administration as well as pulmonal andbronchial administration by inhalation or installation. In someembodiments, the peptide analogue is administered topically to cross theskin.

In one embodiment, the intravenous, subcutaneous and intramuscular formsof parenteral administration are employed.

Local Treatment

In one embodiment, the peptide analogue or composition according to theinvention is used as a local treatment, i.e. is introduced directly tothe site(s) of action. Accordingly, the peptide may be applied to theskin or mucosa directly, or the peptide may be injected into the site ofaction, for example into the diseased tissue or to an end artery leadingdirectly to the diseased tissue.

Pharmaceutical Formulations

In one embodiment the peptide analogues or pharmaceutically acceptablederivatives thereof are administered alone or in combination withpharmaceutically acceptable carriers or excipients, in either single ormultiple doses. The pharmaceutical compositions or compounds accordingto the invention may be formulated with pharmaceutically acceptablecarriers or diluents as well as any other known adjuvants and excipientsin accordance with conventional techniques, such as those disclosed inRemington: The Science and Practice of Pharmacy, 20^(th) Edition,Gennaro, Ed., Mack Publishing Co., Easton, Pa., 2000.

The term “pharmaceutically acceptable derivative” in present contextincludes pharmaceutically acceptable salts, which indicate a salt whichis not harmful to the patient. Such salts include pharmaceuticallyacceptable basic or acid addition salts as well as pharmaceuticallyacceptable metal salts, ammonium salts and alkylated ammonium salts. Apharmaceutically acceptable derivative further includes pharmaceuticallyacceptable esters, prodrugs, or other precursors of a compound which maybe biologically metabolized into the active compound, or crystal formsof a compound.

The pharmaceutical composition or pharmaceutically acceptablecomposition may be specifically formulated for administration by anysuitable route, such as an enteral route, the oral, rectal, nasal,pulmonary, buccal, sublingual, transdermal, intracisternal,intraperitoneal, and parenteral (including subcutaneous, intramuscular,intrathecal, intravenous and intradermal) route.

In an embodiment of the present invention, the pharmaceuticalcompositions or peptides of the present invention are formulated forcrossing the blood-brain-barrier.

Pharmaceutical compositions for oral administration include solid dosageforms such as hard or soft capsules, tablets, troches, dragees, pills,lozenges, powders and granules. Where appropriate, they can be preparedwith coatings such as enteric coatings, or they can be formulated so asto provide controlled release of the active ingredient, such assustained or prolonged release, according to methods well known in theart. In the same solid dosage form two active ingredients may becombined so as to provide controlled release of one active ingredientand immediate release of another active ingredient.

Liquid dosage forms for oral administration include solutions,emulsions, aqueous or oily suspensions, syrups and elixirs.

Pharmaceutical compositions for parenteral administration includesterile aqueous and non-aqueous injectable solutions, dispersions,suspensions or emulsions, as well as sterile powders to be reconstitutedin sterile injectable solutions or dispersions prior to use. Depotinjectable formulations are also regarded as being within the scope ofthe present invention.

Other suitable administration forms include suppositories, sprays,ointments, cremes/lotions, gels, inhalants, dermal patches, implants,etc.

In one embodiment, a compound or peptide for use according to thepresent invention is generally utilized as the free substance or as apharmaceutically derivative such as a pharmaceutically acceptable esteror such as a salt thereof. Examples of the latter are: an acid additionsalt of a compound having a free base functionality, and a base additionsalt of a compound having a free acid functionality. The term“pharmaceutically acceptable salt” refers to a non-toxic salt of acompound for use according to the present invention, which salts aregenerally prepared by reacting a free base with a suitable organic orinorganic acid, or by reacting an acid with a suitable organic orinorganic base. When a compound for use according to the presentinvention contains a free base functionality, such salts are prepared ina conventional manner by treating a solution or suspension of thecompound with a chemical equivalent of a pharmaceutically acceptableacid. When a compound for use according to the present inventioncontains a free acid functionality, such salts are prepared in aconventional manner by treating a solution or suspension of the compoundwith a chemical equivalent of a pharmaceutically acceptable base.Physiologically acceptable salts of a compound with a hydroxy groupinclude the anionic form of the compound in combination with a suitablecation, such as sodium or ammonium ion. Other salts which are notpharmaceutically acceptable may be useful in the preparation ofcompounds of the invention, and these form a further aspect of theinvention. Pharmaceutically acceptable acid addition salts include, butare not limited to, hydrochloride, hydrobromide, hydroiodide, nitrate,sulfate, bisulfate, phosphate, acid phosphate, isonicotinate, acetate,trifluoroacetate, trichloroacetate, lactate, salicylate, citrate,tartrate, pantothenate, bitartrate, ascorbate, succinate, maleate,gentisinate, fumarate, gluconate, glucaronate, saccharate, formate,benzoate, glutamate, methanesulfonate, ethanesulfonate, benzensulfonate,p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.

In one embodiment of the present invention, the peptides of the presentinvention are on crystalline forms, for example co-crystallized forms orhydrates of crystalline forms.

The term “prodrug” refers to peptides that are rapidly transformed invivo to yield the parent compound of the above formulae, for example, byhydrolysis in blood or by metabolism in cells, such as for example thecells of the basal ganglia. A thorough discussion is provided in T.Higuchi and V Stella, “Pro-drugs as Novel Delivery Systems,” Vol. 14 ofthe A.C.S. Symposium Series, and in Bioreversible Carriers in DrugDesign, ed. Edward B. Roche, American Pharmaceutical Association andPergamon Press, 1987, both of which are hereby incorporated byreference. Examples of prodrugs include pharmaceutically acceptable,non-toxic esters of the compounds of the present invention. Esters ofthe compounds of the present invention may be prepared according toconventional methods “March's Advanced Organic Chemistry, 5^(th)Edition”. M. B. Smith & J. March, John Wiley & Sons, 2001.

In one embodiment, for parenteral administration, solutions of peptidesaccording to the present invention in sterile aqueous solution, inaqueous propylene glycol or in sesame or peanut oil are employed.Aqueous solutions should be suitably buffered where appropriate, and theliquid diluent rendered isotonic with, e.g., sufficient saline orglucose. Aqueous solutions are particularly suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration. Thesterile aqueous media to be employed are all readily available bystandard techniques known to those skilled in the art.

Suitable pharmaceutical carriers include inert solid diluents orfillers, sterile aqueous solutions and various organic solvents.Examples of solid carriers are lactose, terra alba, sucrose,cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate,stearic acid and lower alkyl ethers of cellulose. Examples of liquidcarriers are syrup, peanut oil, olive oil, phospholipids, fatty acids,fatty acid amines, polyoxyethylene and water. Moreover, the carrier ordiluent may include any sustained release material known in the art,such as glyceryl monostearate or glyceryl distearate, alone or mixedwith a wax. The pharmaceutical compositions formed by combining thecompounds according to the present invention and the pharmaceuticallyacceptable carriers are then readily administered in a variety of dosageforms suitable for the disclosed routes of administration. Theformulations may conveniently be presented in unit dosage form bymethods known in the art of pharmacy.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units, such as capsules or tablets, whicheach contain a predetermined amount of the active ingredient, and whichmay include a suitable excipient.

Furthermore, the orally available formulations may be in the form of apowder or granules, a solution or suspension in an aqueous ornon-aqueous liquid, or an oil-in-water or water-in-oil liquid emulsion.

Compositions intended for oral use may be prepared according to anyknown method, and such compositions may contain one or more agentsselected from the group consisting of sweetening agents, flavouringagents, colouring agents and preserving agents in order to providepharmaceutically elegant and palatable preparations. Tablets may containthe active ingredient(s) in admixture with non-toxic pharmaceuticallyacceptable excipients which are suitable for the manufacture of tablets.These excipients may, for example, be: inert diluents, such as calciumcarbonate, sodium carbonate, lactose, calcium phosphate or sodiumphosphate; granulating and disintegrating agents, for example cornstarch or alginic acid; binding agents, for example, starch, gelatine oracacia; and lubricating agents, for example magnesium stearate, stearicacid or talc. The tablets may be uncoated or they may be coated by knowntechniques to delay disintegration and absorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate may be employed. They may also becoated by the techniques described in U.S. Pat. Nos. 4,356,108;4,166,452; and 4,265,874, the contents of which are incorporated hereinby reference, to form osmotic therapeutic tablets for controlledrelease.

Formulations for oral use may also be presented as hard gelatinecapsules where the active ingredient is mixed with an inert soliddiluent, for example, calcium carbonate, calcium phosphate or kaolin, ora soft gelatine capsules wherein the active ingredient is mixed withwater or an oil medium, for example peanut oil, liquid paraffin, orolive oil.

Aqueous suspensions may contain the compound for use according to thepresent invention in admixture with excipients suitable for themanufacture of aqueous suspensions. Such excipients are suspendingagents, for example sodium carboxymethylcellulose, methylcellulose,hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia; dispersing or wetting agents may be anaturally-occurring phosphatide such as lecithin, or condensationproducts of an alkylene oxide with fatty acids, for examplepolyoxyethylene stearate, or condensation products of ethylene oxidewith long chain aliphatic alcohols, for example,heptadecaethyleneoxycetanol, or condensation products of ethylene oxidewith partial esters derived from fatty acids and a hexitol such aspolyoxyethylene sorbitol monooleate, or condensation products ofethylene oxide with partial esters derived from fatty acids and hexitolanhydrides, for example polyethylene sorbitan monooleate. The aqueoussuspensions may also contain one or more colouring agents, one or moreflavouring agents, and one or more sweetening agents, such as sucrose orsaccharin.

Oily suspensions may be formulated by suspending the active ingredientin a vegetable oil, for example arachis oil, olive oil, sesame oil orcoconut oil, or in a mineral oil such as a liquid paraffin. The oilysuspensions may contain a thickening agent, for example beeswax, hardparaffin or cetyl alcohol. Sweetening agents such as those set forthabove, and flavouring agents may be added to provide a palatable oralpreparation. These compositions may be preserved by the addition of ananti-oxidant such as ascorbic acid.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.Additional excipients, for example, sweetening, flavouring, andcolouring agents may also be present.

The pharmaceutical compositions comprising peptides for use according tothe present invention may also be in the form of oil-in-water emulsions.The oily phase may be a vegetable oil, for example, olive oil or arachisoil, or a mineral oil, for example a liquid paraffin, or a mixturethereof. Suitable emulsifying agents may be naturally-occurring gums,for example gum acacia or gum tragacanth, naturally-occurringphosphatides, for example soy bean, lecithin, and esters or partialesters derived from fatty acids and hexitol anhydrides, for examplesorbitan monooleate, and condensation products of said partial esterswith ethylene oxide, for example polyoxyethylene sorbitan monooleate.The emulsions may also contain sweetening and flavouring agents.

Syrups and elixirs may be formulated with sweetening agents, for exampleglycerol, propylene glycol, sorbitol or sucrose. Such formulations mayalso contain a demulcent, a preservative and flavouring and colouringagent. The pharmaceutical compositions may be in the form of a sterileinjectable aqueous or oleaginous suspension. This suspension may beformulated according to the known methods using suitable dispersing orwetting agents and suspending agents described above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are water, Ringer's solution,and isotonic sodium chloride solution. In addition, sterile, fixed oilsare conveniently employed as solvent or suspending medium. For thispurpose, any bland fixed oil may be employed using synthetic mono- ordiglycerides. In addition, fatty acids such as oleic acid find use inthe preparation of injectables.

The compositions may also be in the form of suppositories for rectaladministration of the compounds of the invention. These compositions canbe prepared by mixing the compound with a suitable non-irritatingexcipient which is solid at ordinary temperatures but liquid at therectal temperature and will thus melt in the rectum to release the drug.Such materials include, for example, cocoa butter and polyethyleneglycols.

Peptides of the present invention may also be administered in the formof liposome delivery systems, such as small unilamellar vesicles, largeunilamellar vesicles, and multilamellar vesicles. Liposomes may beformed from a variety of phospholipids, such as but not limited tocholesterol, stearylamine or phosphatidylcholines.

In addition, some peptides of the present invention may form solvateswith water or common organic solvents. Such solvates are alsoencompassed within the scope of the invention.

Thus, a further embodiment provides a pharmaceutical compositioncomprising a peptide for use according to the present invention, or apharmaceutically acceptable salt, solvate, or prodrug thereof, and oneor more pharmaceutically acceptable carriers, excipients, or diluents.

The present invention also encompass pharmaceutical compositionscomprising the α-MSH, the γ-MSH analogues, the GLP-1 analogues, theGLP-2 analogues and the analogues of N-terminus of Annexin A1 of thepresent invention, as well as the α-MSH and γ-MSH analogues, the GLP-1analogues, the GLP-2 analogues or the analogues and of N-terminus ofAnnexin A1 of the present invention for use as a medicament.

Specifically, the α-MSH and γ-MSH analogues α-MSH, the γ-MSH analogues,the GLP-1 analogues, the GLP-2 analogues and the analogues of N-terminusof Annexin A1 according to the present invention are potentiallysuitable for use in the treatment of an ischemic and/or inflammatorycondition in the tissue of one or more organs of a mammal, wherein saidtreatment may be prophylactic, ameliorative or curative.

Said ischemic conditions concerned may in one embodiment be due to orcaused by underlying conditions such as stroke, injury, septic shock,systemic hypotension, cardiac arrest due to heart attack, cardiacarrhythmia, atheromatous disease with thrombosis, embolism from theheart or from blood vessel from any organ, vasospasm, aortic aneurysm oraneurisms in other organs, coronary stenosis, myocardial infarction,angina pectoris, pericarditis, myocarditis, myxodemia, or endocarditis.

Further, said ischemic and/or inflammatory condition may in oneembodiment be associated with surgery, such as major surgery, whereinsaid surgery may include cardiothoracic surgery, abdominal surgery,surgery on the aorta and/or other major blood vessels, repair of one ormore cardiac valves, cardiac artery bypass grafting (CABG), surgery onthe aortic root or the aortic branch including the common caroticarteries, and combined cardiac surgery such as valve(s) replacement andCABG and/or aortic root surgery.

Furthermore, said ischemic and/or inflammatory condition may in oneembodiment be associated with organ transplantation, such as solid organtransplantation, including heart transplantation, lung transplantation,combined heart and lung transplantation, liver transplantation andkidney transplantation.

In one embodiment, said ischemic and/or inflammatory condition ispost-surgical systemic inflammatory response syndrome (SIRS) orpost-surgical organ dysfunction, including post-surgical renal failuresuch as acute kidney injury (AKI), neprotoxicity and/or chronic renalfailure (CRF).

In one embodiment, said ischemic and/or inflammatory condition isreperfusion injury.

Also, said ischemic and/or inflammatory condition may be an inflammatorydisease, including but not limited to arthropathy (joint disease),rheumatoid arthritis (RA), gout, inflammatory diseases of thegastrointestinal system, and multiple sclerosis.

In addition to the ischemic and/or inflammatory conditions, the α-MSHand γ-MSH analogues α-MSH, the γ-MSH analogues, the GLP-1 analogues, theGLP-2 analogues and the analogues of N-terminus of Annexin A1 accordingto the present invention are potentially suitable for use in thetreatment of an metabolic condition including Type 1 or Type 2 diabetesmellitus, prediabetic conditions including glucose intolerance, obesity,overweight, metabolic syndrome, gestational diabetes mellitus, ormetabolic disease associated with polycystic ovarian syndrome whereinsaid treatment may be prophylactic, ameliorative or curative.

The modified peptides according to this invention may be used to treatthe same diseases and conditions for which the parent peptide has beenindicated.

EXAMPLES Example 1—Synthesis of BAP-Modified α-MSH Peptide Analogues

α-MSH Analogue 1:

Ac-(Ac-Lys-Lys-)Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH2

γ-MSH analogue 1:

Ac-(Ac-Lys-Lys-)Lys-Tyr-Val-Met-Gly-His-Phe-Arg-(D-Trp)-Asp-Arg-Phe-Gly-NH2.

The peptides are manufactured using Fmoc (9-fluorenylmethyloxycarbonyl)chemistry. Peptides are made using a polystyrene resin, functionalizedwith an appropriate linker, and the peptides are then manufactured usingan Intavis Peptide Synthesizer. A 4-fold excess of amino acid is addedrelative to the resin and either HATU(O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate) or HCTU(2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate) were used at a 3.95-fold excess to create theactive ester. Along with an 8-fold excess of DIPEA(N,N-Diisopropylethylamine) as the base, these reagents catalyze theaddition of the next amino acid. Once the amino acid is coupled (eachcycle includes a double coupling cycle to insure efficient coupling) theresin is exposed to 20% acetic anhydride to terminate (“cap-off”) anypeptide chains that have not added the next amino acid.

The amino acids are dissolved in NMP (N-Methyl-2-pyrrolidone) or DMF(Dimethylformamide) For washing. Piperidine is used to remove the Fmocgroup at the end of each coupling cycle which allows the next amino acidto be added.

α-MSH analogue 1 was made with Lys(Mtt) on the end; the peptide wasacetylated, the Mtt was removed, added Lys, added Lys and then acetylateagain.

For γ-MSH analogue 1 the addition of one or more pseudoproline(oxazolidine) dipeptides during the synthesis of serine- and/orthreonine-containing peptides resulted in improvements in peptidequality and an increase in the yield of full length crude peptide. Inthis case the peptide was made up to the MEHF, a pseudoproline dipeptide(Fmoc-YS) was added, the next amino acid “Ser” was coupled 3 times toinsure it went to completion, and the peptide finished manually byadding the Lys(Mtt), acetylating, and then finishing as above.

In each case the peptides were dried using MeOH (3×), DCM (3×), cleavedusing 92% TFA, 2% water, 2% triisopropylsilane, 2% thioanisole and 2%ethanedithiol for 3-4h at room temperature. Peptides were precipitatedin cold diethyl ether, centrifuged (2,000 RPM) and the pellets washed 2×with cold ether. After drying the peptides were solubilized in watercontaining 0.1% TFA (buffer A) and subjected to RP-HPLC using C18columns (buffer B=95% acetonitrile/0.1% TFA).

The purity was determined by analytical HPLC and theoretical monoisotopic molecular masses we confirmed by MS. The sequence integrity wasverified by CID tandem MS/MS sequencing.

Example 2: Pharmacological Characterization of BAP-Modified α-MSHAnalogues

Method:

Murine B16-F1 cells expressing MCr1 are used for determination ofbinding affinity to and agonist activity against the MC1r. Humanrecombinant CHO cells expressing MC3r, MC4r or MC5r are used fordetermination of affinity to or agonist activity against the MC3r, MC4rand MC5r, respectively. The binding affinity is determined inexperiments conducted as described in the procedures for radioligandbinding study catalogue no 0644 (MC1r); no 0447 (MC3r); no 0420 (MC3r)and no 0448 (Mc5r) Cerep, France. In all experiments the Ki values arecalculated based on the ability of the test compound to displace¹²⁵I-NDP-αMSH. The test items are tested in a concentration range of10⁻¹³ to 10⁻⁵M.

For agonist activity against the MCRs the following procedures areconducted: The cells are incubated with test item at concentrations from10¹³ to 10⁻⁵M. In all assays cAMP accumulation is determined afterincubation and the response at a given concentration is expressed as apercent of the maximal control specific agonist response (measuredspecific response/control specific agonist response)×100) when NDP-αMSHis used as positive control. The EC₅₀ values (concentration producing ahalf-maximal specific response) are determined by non-linear regressionanalysis of the concentration-response curves generated with meanreplicate values using Hill equation curve fitting(Y=D+[(A−D)/(1+(C/C₅₀)_(n)H)], where Y=specific response, D=minimumspecific response, A=maximum specific response, C=compoundconcentration, and C₅₀=EC₅₀, and nH=slope factor). For further detailssee the specific protocols for Cerep study protocol no 2147 (MC1r); no0959 (MC3r), no 0699 (MC4r) and no 1869 (MC5r).

Test Peptide Analogues:

Analogue 1:Ac-(Ac-Lys-Lys)Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂

Analogue 2:Ac-(Ac-Lys-Lys)Lys-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-(D-Val)-NH₂

Analogue 3:Ac-(Ac-Lys-Lys)Lys-Ser-Tyr-Ser-Nle-Glu-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂

Analogue 4:Ac-(Ac-Lys-Lys)Lys-Ser-Ser-Ile-Ile-Ser-His-DPhe-Arg-Trp-Gly-Lys-Pro-Val-NH₂

Control peptide: Control peptide 1 (αMSH): (SEQ ID NO: 1)Ac-Ser-Tyr-Ser-Met-Glu-His-Phe- Arg-Trp-Gly-Lys-Pro-Val- NH₂Control peptide 2 (NDP-αMSH): (SEQ ID NO: 30)Ac-Ser-Tyr-Ser-Nle-Glu-His-DPhe- Arg-Trp-Gly-Lys-Pro-Val-NH₂

Results

Ki values (nM):

MC1r MC3r MC4r MC5r Control peptide 1 4.6 114 128 404 Control peptide 21.5 8.8 7.1 14 Analogue 1 1.2 24 26 193MCr binding relative to control peptide 1:

MC1r MC3r MC4r MC5r Analogue 1   4x  5x  5x 2x Analogue 2 3.3x  8.5x Analogue 3 41x 47x Analogue 4 9.4x 42x 53xEC₅₀ values (nM):

MC1r MC3r MC4r MC5r Control peptide 1 13 62 28 1104 Control peptide 2 1524 10 40 Analogue 1 8.9 53 18 625

Example 3: Pharmacological Characterization of BAP-Modified γ-MSHAnalogues

Test Peptide Analogues:

Analogue 1:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Met-Gly-His-Phe-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂

Analogue 2:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-NH₂

Analogue 3:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Met-Gly-His-DPhe-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂

Analogue 4:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Met-Gly-His-Phe-Arg-DTrp-Asp-Arg-DPhe-Gly-NH₂

Analogue 5:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Nle-Gly-His-Phe-Arg-DTrp-Asp-Arg-Phe-Gly-NH₂

Analogue 6:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Nle-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-Gly-NH₂

Analogue 7:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Nle-Gly-His-DPhe-Arg-Trp-Asp-Arg-Phe-Gly-NH₂

Analogue 8:Ac-(Ac-Lys-Lys)Lys-Tyr-Val-Nle-Gly-His-DPhe-Arg-DTrp-Asp-Arg-Phe-Gly-NH2

Control peptides: Control peptide 1 (y2-MSH): (SEQ ID NO: 2)Ac-Tyr-Val-Met-Gly-His-Phe-Arg-Trp- Asp-Arg-Phe-Gly-NH₂Control peptide 2 (DTrp-y2-MSH): (SEQ ID NO: 59)Ac-Tyr-Val-Met-Gly-His-Phe-Arg-DTrp- Asp-Arg-Phe-Gly-NH₂

Results

Ki values (nM):

MC1r MC3r MC4r MC5r y.2MSH 104 37 328 1022 DTrp-y.2MSH 28 34 173 369Analogue 1 3.8 15 150 215MCr binding affinity relative to control peptide 1:

MC1r MC3r MC4r MC5r Analogue 1 27x 5x 2x 5xMCr binding affinity relative to control peptide 2:

MC1r MC3r MC4r MC5r Analogue 1 4x 2x 2x 3xMCr binding affinity relative to αMSH(Ac-Ser-Tyr-Ser-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro-Val-NH₂) (SEQ ID NO:1):

MC1r MC3r MC4r MC5r Control peptide 1 0.04x  1.5x  0.1x 0.4x Controlpeptide 2 0.16x 0.36x 0.74x 1.1x Analogue 1  1.2x  7.6x  0.9x   2xAnalogue 2  0.5x 11.6x  1.2x Analogue 3   20x  8.5x Analogue 4  7.7x 4.8x  1.3x Analogue 5  1.9x   38x  2.8x Analogue 6  4.6x  2.9x Analogue7   46x   37x Analogue 8  7.3x   37x 10.7xEC₅₀ values (nM):

MC1r MC3r MC4r MC5r Control peptide 1 141 46 279 1787 Control peptide 2167 170 56 505 Analogue 1 68 2.0 252 555

Example 4: Synthesis of BAP Modified Peptides

Similarly the synthesis of BAP modified α-MSH peptide analogues(Example 1) peptides were synthesized using standard Fmoc chemistryusing 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium3-oxide hexafluorophosphate (HATU) or2-(6-Chloro-1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminiumhexafluorophosphate (HCTU) as the coupling reagents with Hunig's Base(N,N-diisopropylethylamine, DIPEA). For the lysine branching asdescribed in more detail below, combination of orthogonally protectedlysines were used including Fmoc-Lys(MTT)-OH, Fmoc-Lys(ivDde)-OH, andFmoc-Lys(Boc)-OH.

Peptides were cleaved with standard cleavage cocktails includingtrifluoroacetic acid, triisoproproylsilane, and water and precipitatedwith ice-cold ether. All crude peptides were purified by reversed-phasechromatography on columns with C-18 functionality and using gradients ofacetonitrile, deionized water, and trifluoroacetic acid as runningbuffers. Purity was determined by high-pressure liquid chromatographyand mass (MS) and sequence (tandem MS) information was obtained using ananospray mass spectrometer.

BAP Attached in the C-Terminus of the Sequence

Branching on the C-terminal lysine (METHOD 1):N-α-Fmoc-N-ε-4-methyltrityl-L-lysine was added to Rink amide resin afterpiperidine deprotection. The remaining sequence of the target peptidewas added and the full length sequence was acetylated with aceticanhydride. The methyltrityl group was then removed using 1%trifluoroacetic acid in dichloromethane. AdditionalNα-Fmoc-Nε-Boc-L-lysine was then added to the side chain and acetylatedwhen desired.

Branching on other than the C-terminal lysine: analogously to attachingBAP to lysines in the sequence between the N- and C-termini (METHOD 2).

BAP Attached to Lysines in the Sequence Between the N- and C-Termini

METHOD 2: N-α-Fmoc-N-ε-4-methyltrityl-L-lysine was added to the peptidesequence, methytrityl was removed after finalizing the sequence andoptionally N-terminal acetylation. Appropriate lysine analogues such asFmoc-Lys(MTT)-OH, Fmoc-Lys(ivDde)-OH and Fmoc-Lys(Boc)-OH weresequentially added and selectively deprotected, before acetylation toensure appropriate side chain and acetyl addition.

BAP Attached in the N-Terminus of the Sequence

Branching on the N-terminal lysine (METHOD 3):N-α-Fmoc-N-ε-4-methyltrityl-L-lysine was added to N-terminal of thesequence, Fmoc was removed, the sequence acetylated at the N-terminus,and the methyltrityl group was removed. AdditionalNα-Fmoc-Nε-Boc-L-lysine was then added to the side chain and acetylatedwhen desired.

Branching on other than the N-terminal lysine: analogously to attachingBAP to lysines in the sequence between the N- and C-termini (METHOD 2).

Peptides

Analogue 1 (by METHOD 1):

Ac-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-(Lys-Lys-Ac)Lys-NH2

Purity: 99.1%

MS: 546.9, 637.8, 765.2

Analogue 2 (by METHOD 1):

Ac-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-Gly-(Lys-Lys-Ac)Lys-NH2

Purity: 99.3%

MS: 635.5, 762.4, 952.7

Analogue 3 (by METHOD 1):

Ac-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-(Lys-Lys-Ac)Lys-NH2

Purity: 96.2%

MS: 605.5, 706.2, 847.2, 1058.8

Analogue 4 (by METHOD 1):

Ac-His-Gly-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-(Lys-Lys-Ac)Lys-NH2

Purity: 95.1%

MS: 603.5, 703.9, 844.4, 1055.3

Analogue 5 (by METHOD 1):

Ac-His-(D-Ala)-Asp-Gly-Ser-Phe-Ser-Asp-Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn-Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-(Lys-Lys-Ac)Lys-NH2

Purity: 96.9%

MS: 706.2, 847.2, 1058.8

Analogue 6 (by METHOD 1):

Ac-Ala-Met-Val-Ser-Glu-Phe-Leu-Lys-Gln-(D-Ala)-Trp-Phe-Ile-Glu-Asn-Glu-Glu-Gln-Glu-Tyr-Val-Gln-Thr-Val-Lys-Lys-(Lys-Ac)Lys-NH2

Purity: 96.0

MS: 879.2

Analogue 7 (by METHOD 2):

Ac-Ala-Met-Val-Ser-Glu-Phe-Leu-(Ac-(Lys-Ac)Lys-Lys)Lys-Gln-Ala-Trp-NH2

Purity: 97.3

MS: 442.2, 662.9

Example 5: Pharmacological Characterization of BAP-Modified GLP-1Analogues

Method:

CHO-K1 cells expressing the human GLP-1 receptor grown in media withoutantibiotic were detached by gentle flushing with PBS-EDTA (5 mM EDTA),recovered by centrifugation and resuspended in assay buffer (KRH: 5 mMKCl, 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mMKH2PO4, 1.45 mM CaCl2, 0.5 g/l BSA).

12 μl of cells were mixed with 12 μl of the test compound (solubilizedin PBS/0.5% BSA and finally diluted from a stock solution of 1 mM) atincreasing concentrations in 96 wells plates and then incubated 30 minat room temperature. cAMP production was determined after addition of alysis buffer and 1 hour incubation, by use of competitive immunoassayusing cryptate-labeled anti-cAMP and d2-labeled cAMP (HTRF kit fromCisBio) with Delta F percentage values calculated according to themanufacturer specification. Dose response curves were performed inparallel with test compounds, and reference compounds.

The HTRF technology is a titration assay based on a competition betweenlabeled cAMP (exogenous) and cAMP produced by the cell after activationof the MCr. The dynamic range of the assay was 3-4 fold meaning that thelinear range (which enables conversion from raw data to nM of cAMP) iswithin that range. The window between top and bottom of the curve ishigher (around 100) which means that converting into nM of cAMP, theassay window of cAMP goes from 1 nM (basal) to around 30 nM (E_(max)).All experiments were conducted in the presence of the non-selectivephosphodiesterase inhibitor IBMX (1 mM in final concentration).

The test compounds were tested in a concentration range from 10¹⁴ to10⁻⁷M

Data is presented as mean values. The EC₅₀ (ie the concentration induced50% of max response) and the hill slope were determined by best fitanalyses after logarithmic transformation using the graph pad software(version 6.0).

Control peptide: GLP-1 (7-36): (SEQ ID NO: 112)H-His-Ala-Glu-Gly-Thr-Phe-Thr-Ser- Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala- Trp-Leu-Val-Ly s-Gly-Arg-Gly-OH

Results:

GLP-1 (7-36) Analogue 1 Analogue 2 Max efficacy in % of 101.3 102.4control peptide EC₅₀ (nM) 0.016 0.016 0.040 Hill Slope 1.28 0.73 0.80

Both analogue 1 and 2 showed full agonist activity when compared to thecontrol peptide GLP-1 (7-36) with sub-nano molar EC₅₀ values comparableto the control peptide GLP-1 (7-37). The lower Hill Slope indicates thatagonist activity can be obtained at lower concentrations than what wouldbe seen with the control peptide.

Example 6: Pharmacological Characterization of BAP-Modified GLP-2Analogues

Method:

CHO-K1 cells expressing the human GLP-2 receptor grown in media withoutantibiotic were detached by gentle flushing with PBS-EDTA (5 mM EDTA),recovered by centrifugation and resuspended in assay buffer (KRH: 5 mMKCl, 1.25 mM MgSO4, 124 mM NaCl, 25 mM HEPES, 13.3 mM Glucose, 1.25 mMKH2PO4, 1.45 mM CaCl₂), 0.5 g/l BSA).

12 μl of cells were mixed with 12 μl of the test compound (solubilizedin PBS/0.5% BSA and finally diluted from a stock solution of 1 mM) atincreasing concentrations in 96 wells plates and then incubated 30 minat room temperature. cAMP production was determined after addition of alysis buffer and 1 hour incubation, by use of competitive immunoassayusing cryptate-labeled anti-cAMP and d2-labeled cAMP (HTRF kit fromCisBio) with Delta F percentage values calculated according to themanufacturer specification. Dose response curves were performed inparallel with test compounds, and reference compounds.

The HTRF technology is a titration assay based on a competition betweenlabeled cAMP (exogenous) and cAMP produced by the cell after activationof the MCr. The dynamic range of the assay was 3-4 fold meaning that thelinear range (which enables conversion from raw data to nM of cAMP) iswithin that range. The window between top and bottom of the curve ishigher (around 100) which means that converting into nM of cAMP, theassay window of cAMP goes from 1 nM (basal) to around 30 nM (E_(max)).All experiments were conducted in the presence of the non-selectivephosphodiesterase inhibitor IBMX (1 mM in final concentration).

The test compounds were tested in a concentration range from 10¹⁴ to10⁻⁷ M

Data is presented as mean values. The EC₅₀ (ie the concentration induced50% of max response) and the Hill slope were determined by best fitanalyses after logarithmic transformation using the graph pad software(version 6.0).

Control peptide: GLP-2 (1-34): (SEQ ID NO: 113)H-His-Ala-Asp-Gly-Ser-Phe-Ser-Asp- Glu-Met-Asn-Thr-Ile-Leu-Asp-Asn-Leu-Ala-Ala-Arg-Asp-Phe-Ile-Asn- Trp-Leu-Ile-Gln-Thr-Lys-Ile-Thr-Asp-Arg-OH

Results:

GLP-2 (1-34) Analogue 3 Analogue 4 Analogue 5 Max efficacy in 101.3101.5 95.1 % of control peptide EC₅₀ (nM) 0.36 3.4 5.9 8.9 Hill Slope0.78 0.97 0.96 1.03

All three analogues showed full agonist activity when compared to thecontrol peptide GLP-2 (1-34) with EC₅₀ values in the nM range.Consequently the current examples of BAP modified GLP-2 analogues arefull and potent agonists against the human GLP-2 receptor.

Example 7: Pharmacological Characterization of BAP-Modified AnxA1N-Terminal Fragments

Method:

Recombinant cells co-expressing mitochondrial apoaequirin andrecombinant human type 2 Formyl peptide receptor (FPR2) were grown for18 hours prior to testing in media without antibiotics were detached bygentle flushing with PBS-EDTA (5 mM EDTA), recovered by centrifugationand re-suspended in assay buffer (DHEM/HAM's F12 with HEPES and 0.1%protease free BSA). Cells were then incubated for at least 4 hours atroom temperature coelenterazine h (from Molecular Probes) before DRstudies were conducted.

50 μl of cell suspension was injected on 50 μl of the test or controlcompound (solubilized in PBS/0.5% BSA and finally diluted from a stocksolution of 1 mM) at increasing concentrations in 96 wells plates andthen incubated 30 min at room temperature. The resulting light emissionwas recorded using the Hamamatsu functional drug Screening system 6000(FDSS6000). For standardization of emission of recorded light acrossplates and between experiments 100 μM digitonin or 20 μM ATP were addedto some of the wells.

Agonist activity was expressed as % of the maximal activity obtainedwith the internal control compound Trp-Lys-Tyr-Met-Val-Met (SEQ ID NO:114)

The test compounds were tested in a concentration range from 10⁻¹¹ to10⁻⁵ M

Data is presented as mean values. When possible the EC₅₀ (ie theconcentration induced 50% of max response) was determined by best fitanalyses after logarithmic transformation using the graph pad software(version 6.0).

Control peptide 1: AnxA1 (2-26): (SEQ ID NO: 115)Ac-Ala-Met-Val-Ser-Glu-Phe-Leu- Lys-Gln-Ala-Trp-Phe-Ile-Glu-Asn-Glu-Glu-Gln-Glu-Tyr-Val-Gln-Thr- Val-Lys-OH Control peptide 2:AnxAl (2-12): (SEQ ID NO: 116) Ac-Ala-Met-Val-Ser-Glu-Phe-Leu-Lys-Gln-Ala-Trp-NH2

Results:

In the literature both the AnxA1 (2-12) and the AnxA1 (2-26)N-terminalfragments of the AnxA1 protein are described as agonist to the FPR2receptor—none of them as significantly potent, but the in the settingneither control peptide 1 or 2 were able to induce agonist activity inthe applied concentration range. In contrast to this both analogue 6 andanalogue 7 showed agonist activity. For Analogue 6 the agonist activityreached 33% of the max response seen with the internal control compoundTrp-Lys-Tyr-Met-Val-Met (SEQ ID NO: 114). The corresponding maximalactivity of control compound 1 was less than 1%. For analogue 7, EC₅₀was determined to 1.56 μM. The maximal agonist response obtained at thehighest tested dose was 85% of the max response obtained with theTrp-Lys-Tyr-Met-Val-Met (SEQ ID NO: 114) hexapeptide, whereas themaximal activity of control peptide was less than 1%.

1-34. (canceled)
 35. A peptide analogue comprising a peptide and one ormore branched amino acid probes, wherein said branched amino acid probecomprises a first amino-alkyl amino acid residue, said first amino-alkylamino acid residue optionally being covalently linked to a secondamino-alkyl amino acid residue, or to a second and a third amino-alkylamino acid residue, to form a linear chain of 2 or 3 amino-alkyl aminoacid residues, wherein the side chain of one or more of said first,second and/or third amino-alkyl amino acid residues are each modified byattaching to the side chain amino group a molecule independentlyselected from the group consisting of AAA_(q)-AAA (SEQ ID NO: 168);(aa₃)_(p)-AAA_(q) (SEQ ID NO: 169-174); AAA_(q)-(aa₃)_(p) (SEQ ID NO:175-180); [(aa₃)-AAA]_(p) (SEQ ID NO: 181) and [AAA-(aa₃)]_(p) (SEQ IDNO: 183 and 184); wherein q is a number selected from 0, 1, 2 and 3; pis a number selected from 1, 2 and 3; AAA is an amino-alkyl amino acidresidue; and (aa₃) is an amino acid residue independently selected fromArg, His, Gly and Ala, wherein said amino-alkyl amino acid residues areoptionally acetylated, wherein said first amino-alkyl amino acid residueis covalently linked to the N-terminus of said peptide, covalentlylinked to the C-terminus of said peptide, and/or attached to the sidechain amino group of an amino-alkyl amino acid residue within saidpeptide, with the proviso that said branched amino acid probe consistsof 2 to 9 amino acid residues.
 36. The peptide analogue according toclaim 35, wherein said peptide is selected from the group consisting ofVIP (Vasoactive Intestinal Peptide; PHM27), PACAP (Pituitary AdenylateCyclase Activating Peptide), Peptide PHI 27 (Peptide HistidineIsoleucine 27), GHRH 1-24 (Growth Hormone Releasing Hormone 1-24),Glucagon, Secretin, glicentin precursor, GIP (gastric inhibitorypeptide), prealbumin or transthyretin (TTR), peptide HI-27 and growthhormone releasing factor (GHRF or GHRH), incretins, glucagon-likepeptide-2 (GLP-2), of somatotrophins (such as somatotropin or growthhormone (GH)), Thyrotrophins (such as Thyroid-stimulating hormone (TSH),Corticotropins (such as Adrenocorticotropic hormone (ACTH), andBeta-endorphin), Lactotrophins (such as Prolactin (PRL), Gonadotropins(such as Luteinizing hormone (LH) and Follicle-stimulating hormone(FSH)), Antidiuretic hormone (ADH or vasopressin) Oxytocin, growthhormone-releasing hormone (GHRH), somatostatin, thyrotropin-releasinghormone (TRH), corticotropin-releasing hormone (CRH)Gonadotropin-Releasing Hormone (GnRH), CREB (cAMP responseelement-binding protein), Lactotripeptides, Isoleucine-Proline-Proline(IPP), Valine-Proline-Proline (VPP), NPY (NeuroPeptide Y), PYY (PeptideYY), APP (Avian Pancreatic Polypeptide) and PPY/PP (PancreaticPolypeptide), Proopiomelanocortin (POMC) peptides (including N-TerminalPeptide of Proopiomelanocortin (NPP, or pro-γ-MSH)), Corticotropin(Adrenocorticotropic Hormone, or ACTH), Corticotropin-like IntermediatePeptide (CLIP), β-Lipotropin (β-LPH), Lipotropin Gamma (γ-LPH), β-MSH,β-Endorphin and [Met]Enkephalin); Enkephalin pentapeptides(Met-enkephalin and Leu-enkephalin), Prodynorphin peptides, dynorphins(dynorphin A, dynorphin B, α-neo-endorphin, β-neo-endorphin, and Bigdynorphin), endorphins (beta-endorphin, Alpha-endorphin,Gamma-endorphin, α-neo-endorphin and 3-neo-endorphin), Adrenorphin,Amidorphin, Leumorphin, Nociceptin, Opiorphin, Spinorphin, kinins,tachykinin neuropeptides (including substance P, kassinin, neurokinin A(NKA), neurokinin B (NKB), eledoisin and physalaemin), Bradykinin,Neuromedins/Bombesin-related peptides (including Neuromedin B (NMB),Neuromedin N, Neuromedin S and Neuromedin U (NmU)); Angiotensin,Bombesin, Calcitonin gene-related peptide (CGRP), α-CGRP, β-CGRP,Carnosine, Cocaine and amphetamine regulated transcript (CART), Deltasleep-inducing peptide (DSIP), FMRFamide, FMRFamide-related peptides(FaRPs), Galanin, Galanin-like peptide (GALP), Gastrin releasing peptide(GRP), Neuropeptide S, Neuropeptide Y, Neurophysins (Neurophysin I andNeurophysin II), Neurotensin, Pancreatic polypeptide, Pituitaryadenylate cyclase activating peptide (PACAP), RVD-Hpα, hemopressin, VGF(VGF nerve growth factor inducible), VGF-derived peptides (TLQP-21),Calcitonin, Amylin (or Islet Amyloid Polypeptide (IAPP)), AGG01,Adrenomedullin (AM), Angiopoietin (Ang), Autocrine motility factor, Bonemorphogenetic proteins (BMPs) (BMP1, BMP2, BMP3, BMP4, BMP5, BMP6, BMP7,BMP8a, BMP8b, BMP10, BMP15), Brain-derived neurotrophic factor (BDNF),Epidermal growth factor (EGF), Erythropoietin (EPO), Fibroblast growthfactor (FGF), Glial cell line-derived neurotrophic factor (GDNF),Granulocyte colony-stimulating factor (G-CSF), Granulocyte macrophagecolony-stimulating factor (GM-CSF), Growth differentiation factor-9(GDF9), Hepatocyte growth factor (HGF), Hepatoma-derived growth factor(HDGF), Insulin-like growth factors (IGF), IGF-1, IGF-2,Migration-stimulating factor, Myostatin (GDF-8), neurotrophins,Neurotrophin-3 (NT-3), Neurotrophin-4 (NT-4), Nerve growth factor (NGF),Platelet-derived growth factor (PDGF), Thrombopoietin (TPO),Transforming growth factor alpha (TGF-α), Transforming growth factorbeta (TGF-β), Tumor necrosis factor-alpha (TNF-α), Vascular endothelialgrowth factor (VEGF), Wnt proteins, Wnt1, Wnt2, Wnt2B, Wnt3, Wnt3A,Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, Wnt8A, Wnt8B, Wnt9A, Wnt9B,Wnt10A, Wnt10B, Wnt11, Wnt16, placental growth factor (PGF), FoetalBovine Somatotrophin (FBS), insulin and insulin-like growth factors,IGF-1, IGF-2, IGF-binding proteins, IGFBP1, IGFBP2, IGFBP3, IGFBP4,IGFBP5, IGFBP6, IGFBP7, Relaxin family peptide hormones, relaxin-like(RLN) peptides, insulin-like (INSL) peptides, RLN1 (relaxin 1), RLN2(relaxin 2), RLN3 (relaxin 3), INSL3 (insulin-like peptide 3, Leydigcell-specific insulin-like peptide), INSL4 (insulin-like peptide 4,early placenta insulin-like peptide, ELIP), INSL5 (insulin-like peptide5), INSL6 (insulin-like peptide 6), Gastrin, gastrin-34, gastrin-17,gastrin-14, pentagastrin, thyroid hormone (T4), Thyrotropin-releasinghormone (TRH), vasopressin, protein hormones, glycoprotein hormones,growth hormone (GH), insulin, LH, FSH, Thyroid-stimulating hormone(thyrotropin, TSH), Angiotensin (AGT), Angiotensin I, Angiotensin II,Angiotensin III, Angiotensin IV, Atrial natriuretic peptide (ANP),NT-proBNP, B-type Natriuretic Peptide (BNP), Atrial natriuretic peptide(ANP), and annexin A-II (annexin II), or variants thereof.
 37. Thepeptide analogue according to claim 35, with the proviso that thepeptide is not glucagon-like peptide-1 (GLP-1), GLP-1 (7-37), GLP-1(7-33), exendin-4, or annexin A1; and/or with the proviso that thepeptide is not α-MSH, or γ-MSH.
 38. The peptide analogue according toclaim 35, wherein said amino-alkyl amino acid residue is an amino acidwith a side chain comprising an amino-alkyl group (—C_(n)H_(2n)NH₂). 39.The peptide analogue according to claim 35, wherein said amino-alkylamino acid residue comprises a side chain amino-alkyl group selectedfrom the group consisting of methylamine (—CH₂NH₂), ethylamine(—C₂H₄NH₂), propylamine (—C₃H₆NH₂), n-butylamine (—C₄H₈NH₂), pentylamine(—C₅H₁₀NH₂), n-hexylamine (—C₆H₁₂NH₂), heptylamine (—C₇H₁₄NH₂),octylamine (—C₈H₁₆NH₂), nonylamine (—C₉H₁₈NH₂), decylamine (—C₁₀H₂₀NH₂),undecylamine (—C₁₁H₂₂NH₂) and dodecylamine (—C₁₂H₂₄NH₂).
 40. The peptideanalogue according to claim 35, wherein the side chain amino group ofsaid amino-alkyl amino acid residue is selected from the groupconsisting of the β-amino group (methylamine); the γ-amino group(ethylamine); the δ-amino group (propylamine), the ε-amino group(n-butylamine); the ζ-amino group (pentylamine); the η-amino group(n-hexylamine); the θ-amino group (heptylamine); the ι-amino group(octylamine); the κ-amino group (nonylamine); the λ-amino group(decylamine); the μ-amino group (undecylamine); and the ν-amino group(dodecylamine).
 41. The peptide analogue according to claim 35, whereinthe branched amino acid probe comprises a first amino-alkyl amino acidresidue, said first amino-alkyl amino acid residue being optionallyacetylated, wherein the side chain amino group of said first amino-alkylamino acid residue is modified by attaching a molecule independentlyselected from the group consisting of AAA_(q)-AAA; (aa₃)_(p)-AAA_(q);AAA_(q)-(aa₃)_(p); [(aa₃)-AAA]_(p) and [AAA-(aa₃)]_(p); wherein q is anumber selected from 0, 1, 2 and 3; p is a number selected from 1, 2 and3; AAA is an amino-alkyl amino acid residue (optionally acetylated); and(aa₃) is an amino acid residue independently selected from Arg, His, Glyand Ala.
 42. The peptide analogue according to claim 35, wherein thebranched amino acid probe comprises a first amino-alkyl amino acidresidue covalently linked to a second amino-alkyl amino acid residue, toform a linear chain of 2 amino-alkyl amino acid residues, said firstand/or second amino-alkyl amino acid residues optionally acetylated,wherein the side chain amino group of said first and/or said secondamino-alkyl amino acid residue is modified by attaching a moleculeindependently selected from the group consisting of AAA_(q)-AAA;(aa₃)_(p)-AAA_(q); AAA_(q)-(aa₃)_(p); [(aa₃)-AAA]_(p) and[AAA-(aa₃)]_(p); wherein q is a number selected from 0, 1, 2 and 3; p isa number selected from 1, 2 and 3; AAA is an amino-alkyl amino acidresidue (optionally acetylated); and (aa₃) is an amino acid residueindependently selected from Arg, His, Gly and Ala.
 43. The peptideanalogue according to claim 35, wherein said amino-alkyl amino acidresidues are individually selected from the group consisting ofL-lysine, D-lysine, L-ornithine and D-ornithine.
 44. The peptideanalogue according to claim 35, wherein each of the first, second and/orthird amino-alkyl amino acids of the branched amino acid probe areindividually selected from the group consisting of L-lysine, D-lysine,L-ornithine and D-ornithine.
 45. The peptide analogue according to claim35, wherein each AAA of the molecules AAA_(q)-AAA; (aa₃)_(p)-AAA_(q);AAA_(q)-(aa₃)_(p); [(aa₃)-AAA]_(p) and [AAA-(aa₃)]_(p) are individuallyselected from the group consisting of L-lysine, D-lysine, L-ornithineand D-ornithine.
 46. The peptide analogue according to claim 35, whereinsaid side chain amino group is individually selected from the δ-aminogroup (ornithine) and the ε-amino group (lysine).
 47. The peptideanalogue according to claim 35, wherein the molecule to be attached tosaid side chain amino group(s) is independently selected from the groupconsisting of Lys_(q)-Lys; (aa₃)_(p)-Lys_(q); Lys_(q)-(aa₃)_(p);[(aa₃)-Lys]_(p); [Lys-(aa₃)]_(p); Orn_(q)-Orn; (aa₃)_(p)-Orn_(q);Orn_(q)-(aa₃)_(p); [(aa₃)-Orn]_(p) and [Orn-(aa₃)]_(p); Orn_(p)-Lys_(p);Lys_(p)-Orn_(p); [Orn-Lys]_(p) and [Lys-Orn]_(p), wherein q is a numberselected from 0, 1, 2 and 3; p is a number selected from 1, 2 and 3;(aa₃) is an amino acid residue independently selected from Arg, His, Glyand Ala; and each of said Lys, Orn and (aa)₃ amino acid residues areoptionally acetylated.
 48. The peptide analogue according to claim 35,wherein the branched amino acid probe consist of 2 to 3 amino acidresidues, such as 3 to 4 amino acid residues, for example 4 to 5 aminoacid residues, such as 5 to 6 amino acid residues, for example 6 to 7amino acid residues, such as 7 to 8 amino acid residues, for example 8to 9 amino acid residues.
 49. The peptide analogue according to claim35, wherein the molecule to be attached to the side chain amino group(s)of one or more of the first, second and/or third amino-alkyl amino acidresidues is selected from the group consisting of Lys, Ac-Lys, Lys-Lys,Ac-Lys-Lys, Lys-Lys-Lys, Ac-Lys-Lys-Lys, Lys-Lys-Lys-Lys,Ac-Lys-Lys-Lys-Lys, Lys-Gly-Lys, Ac-Lys-Gly-Lys, Lys-Lys-Gly,Ac-Lys-Lys-Gly, Lys-Gly, Ac-Lys-Gly, Lys-Ala-Lys, Ac-Lys-Ala-Lys,Lys-Lys-Ala, Ac-Lys-Lys-Ala, Lys-Ala, Ac-Lys-Ala, Lys-His-Lys,Ac-Lys-His-Lys, Lys-Lys-His, Ac-Lys-Lys-His, Lys-His, Ac-Lys-His,Lys-Arg-Lys, Ac-Lys-Arg-Lys, Lys-Lys-Arg, Ac-Lys-Lys-Arg, Lys-Arg andAc-Lys-Arg.
 50. The peptide analogue according to claim 35, wherein thebranched amino acid probe comprises a first amino-alkyl amino acidresidue covalently linked to a second and a third amino-alkyl amino acidresidue to form a linear chain of 3 amino-alkyl amino acid residues,said first, second and/or third amino-alkyl amino acid residuesoptionally acetylated, wherein the side chain amino group of said first,second and/or third amino-alkyl amino acid residues is modified byattaching a molecule independently selected from the group consisting ofAAA_(q)-AAA; (aa₃)_(p)-AAA_(q); AAA_(q)-(aa₃)_(p); [(aa₃)-AAA]_(p) and[AAA-(aa₃)]_(p); wherein q is a number selected from 0, 1, 2 and 3; p isa number selected from 1, 2 and 3; AAA is an amino-alkyl amino acidresidue (optionally acetylated); and (aa₃) is an amino acid residueindependently selected from Arg, His, Gly and Ala.
 51. The peptideanalogue according to claim 35, wherein the side chain of i) one of saidfirst, second and/or third amino-alkyl amino acid residues, ii) two ofsaid first, second and/or third amino-alkyl amino acid residues, or iii)three of said first, second and third amino-alkyl amino acid residues,are modified by attaching a molecule to the side chain amino group. 52.The peptide analogue according to claim 35, wherein said amino-alkylamino acid residues are individually selected from the group consistingof L-lysine and L-ornithine.
 53. The peptide analogue according to claim35, wherein the molecule to be attached to said side chain aminogroup(s) is independently selected from the group consisting ofLys_(q)-Lys; Orn_(q)-Orn; Orn_(p)-Lys_(p); Lys_(p)-Orn_(p);[Orn-Lys]_(p) and [Lys-Orn]_(p), wherein q is a number selected from 0,1, 2 and 3; p is a number selected from 1, 2 and 3; and each of said Lysand Orn amino acid residues are optionally acetylated.
 54. A method oftreating a patient suffering from a disease or condition, said methodcomprising administering to said patient the peptide analogue accordingto claim 35, said disease or condition selected from the groupcomprising stroke, injury, septic shock, systemic hypotension, cardiacarrest due to heart attack, cardiac arrhythmia, atheromatous diseasewith thrombosis, embolism from the heart or from blood vessel from anyorgan, vasospasm, aortic aneurysm or aneurisms in other organs, coronarystenosis, myocardial infarction, angina pectoris, pericarditis,myocarditis, myxodemia, or endocarditis, ischemic and/or inflammatorycondition, be associated with surgery, such as major surgery,cardiothoracic surgery, abdominal surgery, surgery on the aorta and/orother major blood vessels, repair of one or more cardiac valves, cardiacartery bypass grafting (CABG), surgery on the aortic root or the aorticbranch including the common carotic arteries, and combined cardiacsurgery such as valve(s) replacement and CABG and/or aortic rootsurgery, ischemic and/or inflammatory condition may in one embodiment beassociated with organ transplantation, such as solid organtransplantation, including heart transplantation, lung transplantation,combined heart and lung transplantation, liver transplantation andkidney transplantation, post-surgical systemic inflammatory responsesyndrome (SIRS) or post-surgical organ dysfunction, includingpost-surgical renal failure such as acute kidney injury (AKI),neprotoxicity and/or chronic renal failure (CRF), reperfusion injury,arthropathy (joint disease), rheumatoid arthritis (RA), gout,inflammatory diseases of the gastrointestinal system, and multiplesclerosis, diabetes mellitus, prediabetic conditions including glucoseintolerance, obesity, overweight, metabolic syndrome, gestationaldiabetes mellitus, or metabolic disease associated with polycysticovarian syndrome, wherein said treatment may be prophylactic,ameliorative or curative.